Biochemistry_Lippinco

Biochemistry_Lippinco. Oxidation of the heme iron in hemoglobin from Fe2+ to Fe3+ produces methemoglobin, which cannot bind O2. This oxidation may be acquired and caused by the action of certain drugs, such as nitrates, or endogenous products such as reactive oxygen species (see p. 148). The oxidation may also result from congenital defects, for example, a deficiency of NADH-cytochrome b5 reductase (also called NADH-methemoglobin reductase), the enzyme responsible for the conversion of methemoglobin (Fe3+) to hemoglobin (Fe2+), leads to the accumulation of methemoglobin (Fig. 3.22). [Note: The RBC of newborns have approximately half the capacity of those of adults to reduce methemoglobin.] Additionally, rare mutations in the α-or β-globin chain can cause the production of HbM, an abnormal hemoglobin that is resistant to the reductase. The methemoglobinemias are characterized by “chocolate cyanosis” (a blue coloration of the skin and mucous membranes and brown-colored blood) as a result of the dark-colored


Biochemistry_Lippinco

Biochemistry_Lippinco. to the reductase. The methemoglobinemias are characterized by “chocolate cyanosis” (a blue coloration of the skin and mucous membranes and brown-colored blood) as a result of the dark-colored methemoglobin. Symptoms are related to the degree of tissue hypoxia and include anxiety, headache, and dyspnea. In rare cases, coma and death can occur. Treatment is with methylene blue, which is oxidized as Fe3+ is reduced.


Biochemistry_Lippinco

Biochemistry_Lippinco. E. Thalassemias The thalassemias are hereditary hemolytic diseases in which an imbalance occurs in the synthesis of globin chains. As a group, they are the most common single-gene disorders in humans. Normally, synthesis of the αand β-globin chains is coordinated, so that each α-globin chain has a βglobin chain partner. This leads to the formation of α2β2 (HbA). In the thalassemias, the synthesis of either the α-or the β-globin chain is defective, and hemoglobin concentration is reduced. A thalassemia can be caused by a variety of mutations, including entire gene deletions, or substitutions or deletions of one of many nucleotides in the DNA. [Note: Each thalassemia can be classified as either a disorder in which no globin chains are produced (α0-or β0-thalassemia), or one in which some chains are synthesized but at a reduced level (α+-or β+-thalassemia).] 1.


Biochemistry_Lippinco

Biochemistry_Lippinco. β-Thalassemias: In these disorders, synthesis of β-globin chains is decreased or absent, typically as a result of point mutations that affect the production of functional mRNA. However, α-globin chain synthesis is normal. Excess α-globin chains cannot form stable tetramers and so precipitate, causing the premature death of cells initially destined to become mature RBC. Increase in α2δ2 (HbA2) and α2γ2 (HbF) also occurs. There are only two copies of the β-globin gene in each cell (one on each chromosome 11). Therefore, individuals with β-globin gene defects have either β-thalassemia trait (β-thalassemia minor) if they have only one defective β-globin gene or β-thalassemia major (Cooley anemia) if both genes are defective (Fig. 3.23). Because the β-globin gene is not expressed until late in prenatal development, the physical manifestations of β-thalassemias appear only several months after birth. Those individuals with β-thalassemia minor make some β chains and usually do not require


Biochemistry_Lippinco

Biochemistry_Lippinco. in prenatal development, the physical manifestations of β-thalassemias appear only several months after birth. Those individuals with β-thalassemia minor make some β chains and usually do not require specific treatment. However, those infants born with β-thalassemia major are seemingly healthy at birth but become severely anemic, usually during the first or second year of life, due to ineffective erythropoiesis. Skeletal changes as a result of extramedullary hematopoiesis also are seen. These patients require regular transfusions of blood. [Note: Although this treatment is lifesaving, the cumulative effect of the transfusions is iron overload. Use of iron chelation therapy has improved morbidity and mortality.] The only curative option available is hematopoietic stem cell transplantation.


Biochemistry_Lippinco

Biochemistry_Lippinco. 2. α-Thalassemias: In these disorders, synthesis of α-globin chains is decreased or absent, typically as a result of deletional mutations. Because each individual’s genome contains four copies of the α-globin gene (two on each chromosome 16), there are several levels of α-globin chain deficiencies (Fig. 3.24). If one of the four genes is defective, the individual is termed a “silent” carrier of α-thalassemia, because no physical manifestations of the disease occur. If two α-globin genes are defective, the individual is designated as having α-thalassemia trait. If three α-globin genes are defective, the individual has hemoglobin H (β4) disease, a hemolytic anemia of variable severity. If all four α-globin genes are defective, hemoglobin Bart (γ4) disease with hydrops fetalis and fetal death results, because α-globin chains are required for the synthesis of HbF. [Note: Heterozygote advantage against malaria is seen in both α-and β-thalassemias.] V. CHAPTER SUMMARY


Biochemistry_Lippinco

Biochemistry_Lippinco. Hemoglobin A (HbA), the major hemoglobin in adults, is composed of four polypeptide chains (two α chains and two β chains, α2β2) held together by noncovalent interactions (Fig. 3.25). The subunits occupy different relative positions in deoxyhemoglobin compared with oxyhemoglobin. The deoxy form of Hb is called the “T,” or taut (tense), conformation. It has a constrained structure that limits the movement of the polypeptide chains. The T form is the low-oxygen-affinity form of Hb. The binding of oxygen (O2) to the heme iron causes rupture of some of the ionic and hydrogen bonds and movement of the dimers. This leads to a structure called the “R,” or relaxed, conformation. The R form is the high-oxygen-affinity form of Hb. The oxygen-dissociation curve for Hb is sigmoidal in shape (in contrast to that of myoglobin, which is hyperbolic), indicating that the subunits cooperate in binding O2. The binding of an oxygen molecule at one heme group increases the oxygen affinity of the remaining


Biochemistry_Lippinco

Biochemistry_Lippinco. to that of myoglobin, which is hyperbolic), indicating that the subunits cooperate in binding O2. The binding of an oxygen molecule at one heme group increases the oxygen affinity of the remaining heme groups in the same Hb molecule (cooperativity). Hb’s ability to bind O2 reversibly is affected by the partial pressure of oxygen (pO2), the pH of the environment, the partial pressure of carbon dioxide (pCO2), and the availability of 2,3-bisphosphoglycerate (2,3-BPG). For example, the release of O2 from Hb is enhanced when the pH is lowered or the pCO2 is increased (the Bohr effect), such as in exercising muscle, and the oxygen-dissociation curve of Hb is shifted to the right. To cope long-term with the effects of chronic hypoxia or anemia, the concentration of 2,3-BPG in red blood cells increases. 2,3-BPG binds to the Hb and decreases its oxygen affinity. It therefore also shifts the oxygen-dissociation curve to the right. Fetal hemoglobin (HbF) binds 2,3-BPG less tightly than does HbA


Biochemistry_Lippinco

Biochemistry_Lippinco. 2,3-BPG binds to the Hb and decreases its oxygen affinity. It therefore also shifts the oxygen-dissociation curve to the right. Fetal hemoglobin (HbF) binds 2,3-BPG less tightly than does HbA and has a higher oxygen affinity. Carbon monoxide (CO) binds tightly (but reversibly) to the Hb iron, forming carboxyhemoglobin. Hemoglobinopathies are disorders primarily caused either by production of a structurally abnormal Hb molecule as in sickle cell anemia or synthesis of insufficient quantities of normal Hb subunits as in the thalassemias (Fig. 3.26).


Biochemistry_Lippinco

Biochemistry_Lippinco. Choose the ONE best answer. .1. Which one of the following statements concerning the hemoglobins is correct? A. HbA is the most abundant hemoglobin in normal adults. B. Fetal blood has a lower affinity for oxygen than does adult blood because HbF has an increased affinity for 2,3-bisphosphoglycerate. C. The globin chain composition of HbF is α2δ2. D. HbA1c differs from HbA by a single, genetically determined amino acid substitution. E. HbA2 appears early in fetal life.


Biochemistry_Lippinco

Biochemistry_Lippinco. C. The globin chain composition of HbF is α2δ2. D. HbA1c differs from HbA by a single, genetically determined amino acid substitution. E. HbA2 appears early in fetal life. Correct answer = A. HbA accounts for over 90% of the hemoglobin in a normal adult. If HbA1c is included, the percentage rises to ~97%. Because 2,3bisphosphoglycerate (2,3-BPG) reduces the affinity of hemoglobin for oxygen, the weaker interaction between 2,3-BPG and HbF results in a higher oxygen affinity for HbF relative to HbA. HbF consists of α2γ2. HbA1c is a glycated form of HbA, formed nonenzymically in red blood cells. HbA2 is a minor component of normal adult hemoglobin, first appearing shortly before birth and rising to adult levels (~2% of the total hemoglobin) by age 6 months. .2. Which one of the following statements concerning the ability of acidosis to precipitate a crisis in sickle cell anemia is correct? A. Acidosis decreases the solubility of HbS.


Biochemistry_Lippinco

Biochemistry_Lippinco. .2. Which one of the following statements concerning the ability of acidosis to precipitate a crisis in sickle cell anemia is correct? A. Acidosis decreases the solubility of HbS. B. Acidosis increases the oxygen affinity of hemoglobin. C. Acidosis favors the conversion of hemoglobin from the taut to the relaxed conformation. D. Acidosis shifts the oxygen-dissociation curve to the left. E. Acidosis decreases the ability of 2,3-bisphosphoglycerate to bind to hemoglobin. Correct answer = A. HbS is significantly less soluble in the deoxygenated form, compared with oxyhemoglobin S. Decreased pH (acidosis) causes the oxygen-dissociation curve to shift to the right, indicating decreased oxygen affinity (increased delivery). This favors the formation of the deoxy, or taut, form of hemoglobin and can precipitate a sickle cell crisis. The binding of 2,3bisphosphoglycerate is increased, because it binds only to the deoxy form of hemoglobin.


Biochemistry_Lippinco

Biochemistry_Lippinco. .3. Which one of the following statements concerning the binding of oxygen by hemoglobin is correct? A. The Bohr effect results in a lower oxygen affinity at higher pH values. B. Carbon dioxide increases the oxygen affinity of hemoglobin by binding to the C-terminal groups of the polypeptide chains. C. The oxygen affinity of hemoglobin increases as the percentage saturation increases. D. The hemoglobin tetramer binds four molecules of 2,3bisphosphoglycerate. E. Oxyhemoglobin and deoxyhemoglobin have the same affinity for protons.


Biochemistry_Lippinco

Biochemistry_Lippinco. D. The hemoglobin tetramer binds four molecules of 2,3bisphosphoglycerate. E. Oxyhemoglobin and deoxyhemoglobin have the same affinity for protons. Correct answer = C. The binding of oxygen at one heme group increases the oxygen affinity of the remaining heme groups in the same molecule. A rise in pH results in increased oxygen affinity. Carbon dioxide decreases oxygen affinity because it lowers the pH. Moreover, binding of carbon dioxide to the N-termini stabilizes the taut, deoxy form. Hemoglobin binds one molecule of 2,3-bisphosphoglycerate. Deoxyhemoglobin has a greater affinity for protons than does oxyhemoglobin. .4. β-Lysine 82 in HbA is important for the binding of 2,3-bisphosphoglycerate. In Hb Helsinki, this amino acid has been replaced by methionine. Which of the following should be true concerning Hb Helsinki? A. It should be stabilized in the taut, rather than the relaxed, form.


Biochemistry_Lippinco

Biochemistry_Lippinco. A. It should be stabilized in the taut, rather than the relaxed, form. B. It should have increased oxygen affinity and, consequently, decreased oxygen delivery to tissues. C. Its oxygen-dissociation curve should be shifted to the right relative to HbA. D. It results in anemia. Correct answer = B. Substitution of lysine by methionine decreases the ability of negatively charged phosphate groups in 2,3-bisphosphoglycerate (2,3-BPG) to bind the β subunits of hemoglobin. Because 2,3-BPG decreases the oxygen affinity of hemoglobin, a reduction in 2,3-BPG should result in increased oxygen affinity and decreased oxygen (O2) delivery to tissues. The relaxed form is the high-oxygen-affinity form of hemoglobin. Increased oxygen affinity (decreased delivery) results in a left shift in the oxygen-dissociation curve. Decreased delivery of O2 is compensated for by increased RBC production.


Biochemistry_Lippinco

Biochemistry_Lippinco. .5. A 67-year-old man presented to the emergency department with a 1-week history of angina and shortness of breath. He complained that his face and extremities had taken on a blue color. His medical history included chronic stable angina treated with isosorbide dinitrate and nitroglycerin. Blood obtained for analysis was brown. Which one of the following is the most likely diagnosis? A. Carboxyhemoglobinemia B. Hemoglobin SC disease C. Methemoglobinemia D. Sickle cell anemia E. β-Thalassemia


Biochemistry_Lippinco

Biochemistry_Lippinco. A. Carboxyhemoglobinemia B. Hemoglobin SC disease C. Methemoglobinemia D. Sickle cell anemia E. β-Thalassemia Correct answer = C. Oxidation of the ferrous (Fe2+) iron to the ferric (Fe3+) state in the heme prosthetic group of hemoglobin forms methemoglobin. This may be caused by the action of certain drugs such as nitrates. The methemoglobinemias are characterized by chocolate cyanosis (a blue coloration of the skin and mucous membranes and chocolate-colored blood) as a result of the dark-colored methemoglobin. Symptoms are related to tissue hypoxia and include anxiety, headache, and dyspnea. In rare cases, coma and death can occur. [Note: Benzocaine, an aromatic amine used as a topical anesthetic, is a cause of acquired methemoglobinemia.] .6. Why is hemoglobin C disease a nonsickling disease? In HbC, the polar glutamate is replaced by polar lysine rather than by nonpolar valine as in HbS.


Biochemistry_Lippinco

Biochemistry_Lippinco. In HbC, the polar glutamate is replaced by polar lysine rather than by nonpolar valine as in HbS. .7. What would be true about the extent of red blood cell sickling in individuals with HbS and hereditary persistence of HbF? It would be decreased because HbF reduces HbS concentration. It also inhibits polymerization of deoxy HbS. For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW


Biochemistry_Lippinco

Biochemistry_Lippinco. For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW Collagen and elastin are examples of common, well-characterized fibrous proteins of the extracellular matrix (ECM) that serve structural functions in the body. For example, collagen and elastin are found as components of skin, connective tissue, blood vessel walls, and the sclera and cornea of the eye. Each fibrous protein exhibits special mechanical properties, resulting from its unique structure, which is obtained by combining specific amino acids into regular, secondary structural elements. This is in contrast to globular proteins (discussed in Chapter 3), whose shapes are the result of complex interactions between secondary, tertiary, and, sometimes, quaternary structural elements. II. COLLAGEN


Biochemistry_Lippinco

Biochemistry_Lippinco. II. COLLAGEN Collagen is the most abundant protein in the human body. A typical collagen molecule is a long, rigid structure in which three polypeptides (referred to as α chains) are wound around one another in a rope-like triple helix (Fig. 4.1). Although these molecules are found throughout the body, their types and organization are dictated by the structural role collagen plays in a particular organ. In some tissues, collagen may be dispersed as a gel that gives support to the structure, as in the ECM or the vitreous humor of the eye. In other tissues, collagen may be bundled in tight, parallel fibers that provide great strength, as in tendons. In the cornea of the eye, collagen is stacked so as to transmit light with a minimum of scattering. Collagen of bone occurs as fibers arranged at an angle to each other so as to resist mechanical shear from any direction. A. Types


Biochemistry_Lippinco

Biochemistry_Lippinco. A. Types The collagen superfamily of proteins includes >25 collagen types as well as additional proteins that have collagen-like domains. The three polypeptide α chains are held together by interchain hydrogen bonds. Variations in the amino acid sequence of the α chains result in structural components that are about the same size (~1,000 amino acids long) but with slightly different properties. These α chains are combined to form the various types of collagen found in the tissues. For example, the most common collagen, type I, contains two chains called α1 and one chain called α2 (α12α2), whereas type II collagen contains three α1 chains (α13). The collagens can be organized into three groups, based on their location and functions in the body (Fig. 4.2). 1.


Biochemistry_Lippinco

Biochemistry_Lippinco. 1. Fibril-forming collagens: Types I, II, and III are the fibrillar collagens and have the rope-like structure described above for a typical collagen molecule. In the electron microscope, these linear polymers of fibrils have characteristic banding patterns, reflecting the regular staggered packing of the individual collagen molecules in the fibril (Fig. 4.3). Type I collagen fibers (composed of collagen fibrils) are found in supporting elements of high tensile strength (for example, tendons and corneas), whereas fibers formed from type II collagen molecules are restricted to cartilaginous structures. The fibers derived from type III collagen are prevalent in more distensible tissues such as blood vessels. 2. Network-forming collagens: Types IV and VIII form a three-dimensional mesh, rather than distinct fibrils (Fig. 4.4). For example, type IV molecules assemble into a sheet or meshwork that constitutes a major part of basement membranes.


Biochemistry_Lippinco

Biochemistry_Lippinco. Basement membranes are thin, sheet-like structures that provide mechanical support for adjacent cells and function as a semipermeable filtration barrier to macromolecules in organs such as the kidney and the lung. 3. Fibril-associated collagens: Types IX and XII bind to the surface of collagen fibrils, linking these fibrils to one another and to other components in the ECM (see Fig. 4.2). B. Structure Unlike most globular proteins that are folded into compact structures, collagen, a fibrous protein, has an elongated, triple-helical structure that is stabilized by interchain hydrogen bonds. 1.


Biochemistry_Lippinco

Biochemistry_Lippinco. 1. Amino acid sequence: Collagen is rich in proline and glycine, both of which are important in the formation of the triple-stranded helix. Proline facilitates the formation of the helical conformation of each α chain because its ring structure causes “kinks” in the peptide chain. [Note: The presence of proline dictates that the helical conformation of the α chain cannot be an α helix (see p. 16).] Glycine, the smallest amino acid, is found in every third position of the polypeptide chain. It fits into the restricted spaces where the three chains of the helix come together. The glycine residues are part of a repeating sequence, −Gly–X–Y–, where X is frequently proline, and Y is often hydroxyproline (but can be hydroxylysine, Fig. 4.5). Thus, most of the α chain can be regarded as a polytripeptide whose sequence can be represented as (−Gly–Pro– Hyp–)333. 2.


Biochemistry_Lippinco

Biochemistry_Lippinco. 2. Hydroxyproline and hydroxylysine: Collagen contains hydroxyproline and hydroxylysine, which are nonstandard amino acids (see p. 1) not present in most other proteins. They result from the hydroxylation of some of the proline and lysine residues after their incorporation into polypeptide chains (Fig. 4.6). Therefore, the hydroxylation is a posttranslational modification (see p. 460). [Note: Generation of hydroxyproline maximizes formation of interchain hydrogen bonds that stabilize the triple-helical structure.] 3. Glycosylation: The hydroxyl group of the hydroxylysine residues of collagen may be enzymatically glycosylated. Most commonly, glucose and galactose are sequentially attached to the polypeptide chain prior to triple-helix formation (Fig. 4.7). C. Biosynthesis


Biochemistry_Lippinco

Biochemistry_Lippinco. C. Biosynthesis The polypeptide precursors of the collagen molecule are synthesized in fibroblasts (or in the related osteoblasts of bone and chondroblasts of cartilage). They are enzymically modified and form the triple helix, which gets secreted into the ECM. After additional enzymic modification, the mature extracellular collagen fibrils aggregate and become cross-linked to form collagen fibers. 1.


Biochemistry_Lippinco

Biochemistry_Lippinco. 1. Pro-α chain formation: Collagen is one of many proteins that normally function outside of cells. Like most proteins produced for export, the newly synthesized polypeptide precursors of α chains (prepro-α chains) contain a special amino acid sequence at their amino (N)-terminal ends. This sequence acts as a signal that, in the absence of additional signals, targets the polypeptide being synthesized for secretion from the cell. The signal sequence facilitates the binding of ribosomes to the rough endoplasmic reticulum (RER) and directs the passage of the prepro-α chain into the lumen of the RER. The signal sequence is rapidly cleaved in the lumen to yield a precursor of collagen called a pro-α chain (see Fig. 4.7). 2.


Biochemistry_Lippinco

Biochemistry_Lippinco. Hydroxylation: The pro-α chains are processed by a number of enzymic steps within the lumen of the RER while the polypeptides are still being synthesized (see Fig. 4.7). Proline and lysine residues found in the Y-position of the –Gly–X–Y– sequence can be hydroxylated to form hydroxyproline and hydroxylysine residues. These hydroxylation reactions require molecular oxygen, ferrous iron (Fe2+), and the reducing agent vitamin C (ascorbic acid, see p. 381), without which the hydroxylating enzymes, prolyl hydroxylase and lysyl hydroxylase, are unable to function (see Fig. 4.6). In the case of ascorbic acid deficiency (and, therefore, a lack of proline and lysine hydroxylation), interchain H-bond formation is impaired, as is formation of a stable triple helix. Additionally, collagen fibrils cannot be cross-linked (see 7. below), greatly decreasing the tensile strength of the assembled fiber. The resulting deficiency disease is known as scurvy. Patients with scurvy often show ecchymoses


Biochemistry_Lippinco

Biochemistry_Lippinco. cannot be cross-linked (see 7. below), greatly decreasing the tensile strength of the assembled fiber. The resulting deficiency disease is known as scurvy. Patients with scurvy often show ecchymoses (bruise-like discolorations) on the limbs as a result of subcutaneous extravasation (leakage) of blood due to capillary fragility (Fig. 4.8).


Biochemistry_Lippinco

Biochemistry_Lippinco. 3. Glycosylation: Some hydroxylysine residues are modified by glycosylation with glucose or glucosyl-galactose (see Fig. 4.7). 4. Assembly and secretion: After hydroxylation and glycosylation, three pro-α chains form procollagen, a precursor of collagen that has a central region of triple helix flanked by the nonhelical N-and carboxyl (C)terminal extensions called propeptides (see Fig. 4.7). The formation of procollagen begins with formation of interchain disulfide bonds between the C-terminal extensions of the pro-α chains. This brings the three α chains into an alignment favorable for triple helix formation. The procollagen molecules move through the Golgi apparatus, where they are packaged in secretory vesicles. The vesicles fuse with the cell membrane, causing the release of procollagen molecules into the extracellular space. 5.


Biochemistry_Lippinco

Biochemistry_Lippinco. 5. Extracellular cleavage of procollagen molecules: After their release, the triple-helical procollagen molecules are cleaved by N-and Cprocollagen peptidases, which remove the terminal propeptides, producing tropocollagen molecules. 6. Collagen fibril formation: Tropocollagen molecules spontaneously associate to form collagen fibrils. They form an ordered, parallel array, with adjacent collagen molecules arranged in a staggered pattern, each overlapping its neighbor by a length approximately three quarters of a molecule (see Fig. 4.7). 7.


Biochemistry_Lippinco

Biochemistry_Lippinco. 7. Cross-link formation: The fibrillar array of collagen molecules serves as a substrate for lysyl oxidase. This copper-containing extracellular enzyme oxidatively deaminates some of the lysine and hydroxylysine residues in collagen. The reactive aldehydes that result (allysine and hydroxyallysine) can spontaneously condense with lysine or hydroxylysine residues in neighboring collagen molecules to form covalent cross-links and, thus, mature collagen fibers (Fig. 4.9). [Note: Cross-links can form between two allysine residues.] peroxide. Lysyl oxidase is one of several copper-containing enzymes. Others include ceruloplasmin (see p. 404), cytochrome c oxidase (see p. 76), dopamine hydroxylase (see p. 286), superoxide dismutase (see p. 148), and tyrosinase (see p. 273). Disruption in copper homeostasis causes copper deficiency (X-linked Menkes syndrome) or overload (Wilson disease) (see p. 402). D. Degradation


Biochemistry_Lippinco

Biochemistry_Lippinco. D. Degradation Normal collagens are highly stable molecules, having half-lives as long as several years. However, connective tissue is dynamic and is constantly being remodeled, often in response to growth or injury of the tissue. Breakdown of collagen fibers is dependent on the proteolytic action of collagenases, which are part of a large family of matrix metalloproteinases. For type I collagen, the cleavage site is specific, generating three-quarter and one-quarter length fragments. These fragments are further degraded by other matrix proteinases. E. Collagenopathies


Biochemistry_Lippinco

Biochemistry_Lippinco. E. Collagenopathies Defects in any one of the many steps in collagen fiber synthesis can result in a genetic disease involving an inability of collagen to form fibers properly and, therefore, an inability to provide tissues with the needed tensile strength normally provided by collagen. More than 1,000 mutations have been identified in 23 genes coding for 13 of the collagen types. The following are examples of diseases (collagenopathies) that are the result of defective collagen synthesis.


Biochemistry_Lippinco

Biochemistry_Lippinco. 1. Ehlers-Danlos syndrome: Ehlers-Danlos syndrome (EDS) is a heterogeneous group of connective tissue disorders that result from heritable defects in the metabolism of fibrillar collagen molecules. EDS can be caused by a deficiency of collagen-processing enzymes (for example, lysyl hydroxylase or N-procollagen peptidase) or from mutations in the amino acid sequences of collagen types I, III, and V. The classic form of EDS, caused by defects in type V collagen, is characterized by skin extensibility and fragility and joint hypermobility (Fig. 4.10). The vascular form, due to defects in type III collagen, is the most serious form of EDS because it is associated with potentially lethal arterial rupture. [Note: The classic and vascular forms show autosomaldominant inheritance.] Collagen that contains mutant chains may have altered structure, secretion, or distribution, and it frequently is degraded. [Note: Incorporation of just one mutant chain may result in degradation of the triple


Biochemistry_Lippinco

Biochemistry_Lippinco. contains mutant chains may have altered structure, secretion, or distribution, and it frequently is degraded. [Note: Incorporation of just one mutant chain may result in degradation of the triple helix. This is known as a dominant-negative effect.] 2. Osteogenesis imperfecta: This syndrome, known as “brittle bone disease,” is a genetic disorder of bone fragility characterized by bones that fracture easily, with minor or no trauma (Fig. 4.11). Over 80% of cases of osteogenesis imperfecta (OI) are caused by dominant mutations to the genes that encode the α1 or α2 chains in type I collagen. The most common mutations cause the replacement of glycine (in –Gly–X–Y–) by amino acids with bulky side chains. The resultant structurally abnormal α chains prevent the formation of the required triple-helical conformation. Phenotypic severity ranges from mild to lethal. Type I OI, the most common form, is characterized by mild bone fragility, hearing loss, and blue sclerae. Type II, the most severe


Biochemistry_Lippinco

Biochemistry_Lippinco. conformation. Phenotypic severity ranges from mild to lethal. Type I OI, the most common form, is characterized by mild bone fragility, hearing loss, and blue sclerae. Type II, the most severe form, is typically lethal in the perinatal period as a result of pulmonary complications. In utero fractures are seen (see Fig. 4.11). Type III is also a severe form and is characterized by multiple fractures at birth, short stature, spinal curvature leading to a humped-back (kyphotic) appearance, and blue sclerae. Dentinogenesis imperfecta, a disorder of tooth development, may be seen in OI.


Biochemistry_Lippinco

Biochemistry_Lippinco. III. ELASTIN In contrast to collagen, which forms fibers that are tough and have high tensile strength, elastin is a connective tissue fibrous protein with rubber-like properties. Elastic fibers composed of elastin and glycoprotein microfibrils are found in the lungs, the walls of large arteries, and elastic ligaments. They can be stretched to several times their normal length but recoil to their original shape when the stretching force is relaxed. A. Structure


Biochemistry_Lippinco

Biochemistry_Lippinco. Elastin is an insoluble protein polymer generated from a precursor, tropoelastin, which is a soluble polypeptide composed of ~700 amino acids that are primarily small and nonpolar (for example, glycine, alanine, and valine). Elastin is also rich in proline and lysine but contains scant hydroxyproline and hydroxylysine. Tropoelastin is secreted by the cell into the ECM. There, it interacts with specific glycoprotein microfibrils, such as fibrillin, which function as a scaffold onto which tropoelastin is deposited. Some of the lysyl side chains of the tropoelastin polypeptides are oxidatively deaminated by lysyl oxidase, forming allysine residues. Three of the allysyl side chains plus one unaltered lysyl side chain from the same or neighboring polypeptides form a desmosine cross-link (Fig. 4.12). This produces elastin, an extensively interconnected, rubbery network that can stretch and bend in any direction when stressed, giving connective tissue elasticity (Fig. 4.13). Mutations in the


Biochemistry_Lippinco

Biochemistry_Lippinco. This produces elastin, an extensively interconnected, rubbery network that can stretch and bend in any direction when stressed, giving connective tissue elasticity (Fig. 4.13). Mutations in the fibrillin-1 protein are responsible for Marfan syndrome, a connective tissue disorder characterized by impaired structural integrity in the skeleton, the eye, and the cardiovascular system. With this disease, abnormal fibrillin protein is incorporated into microfibrils along with normal fibrillin, inhibiting the formation of functional microfibrils. [Note: Patients with Marfan syndrome, OI, or EDS may have blue sclerae due to tissue thinning that allows underlying pigment to show through.]


Biochemistry_Lippinco

Biochemistry_Lippinco. B. α1-Antitrypsin in elastin degradation Blood and other body fluids contain a protein, α1-antitrypsin (AAT), which inhibits a number of proteolytic enzymes (called peptidases, proteases, or proteinases) that hydrolyze and destroy proteins. [Note: The inhibitor was originally named AAT because it inhibits the activity of trypsin, a proteolytic enzyme synthesized as trypsinogen by the pancreas (see p. 248).] AAT has the important physiologic role of inhibiting neutrophil elastase, a powerful protease that is released into the extracellular space and degrades elastin of alveolar walls as well as other structural proteins in a variety of tissues (Fig. 4.14). Most of the AAT found in plasma is synthesized and secreted by the liver. Extrahepatic synthesis also occurs. 1.


Biochemistry_Lippinco

Biochemistry_Lippinco. 1. α1-Antitrypsin in the lungs: In the normal lung, the alveoli are chronically exposed to low levels of neutrophil elastase released from activated and degenerating neutrophils. The proteolytic activity of elastase can destroy the elastin in alveolar walls if unopposed by the action of AAT, the most important inhibitor of neutrophil elastase (see Fig. 4.14). Because lung tissue cannot regenerate, the destruction of the connective tissue of alveolar walls caused by an imbalance between the protease and its inhibitor results in pulmonary disease. 2.


Biochemistry_Lippinco

Biochemistry_Lippinco. α1-Antitrypsin deficiency and emphysema: In the United States, ~2%– 5% of patients with emphysema are predisposed to the disease by inherited defects in AAT. A number of different mutations in the gene for AAT are known to cause a deficiency of the protein, but one single purine base mutation (GAG to AAG, resulting in the substitution of lysine for glutamic acid at position 342 of the protein) is clinically the most widespread and severe. [Note: The mutated protein is termed the Z variant.] The mutation causes the normally monomeric AAT to misfold, polymerize, and aggregate within the RER of hepatocytes, resulting in decreased secretion of AAT by the liver. AAT deficiency is, therefore, a misfolded protein disease. Consequently, blood levels of AAT are reduced, decreasing the amount that gets to the lung. The polymer that accumulates in the liver may result in cirrhosis (scarring of the liver). In the United States, the AAT mutation is most common in Caucasians of Northern European


Biochemistry_Lippinco

Biochemistry_Lippinco. gets to the lung. The polymer that accumulates in the liver may result in cirrhosis (scarring of the liver). In the United States, the AAT mutation is most common in Caucasians of Northern European ancestry. An individual must inherit two abnormal AAT alleles to be at risk for the development of emphysema. In a heterozygote, with one normal and one defective gene, the levels of AAT are sufficient to protect the alveoli from damage. [Note: Methionine 358 in AAT is required for the binding of the inhibitor to its target proteases. Smoking causes the oxidation and subsequent inactivation of the methionine, thereby rendering the inhibitor powerless to neutralize elastase. Smokers with AAT deficiency, therefore, have a considerably elevated rate of lung destruction and a poorer survival rate than nonsmokers with the deficiency.] The deficiency of elastase inhibitor can be treated by weekly augmentation therapy, that is, intravenous administration of AAT. The AAT diffuses from the blood


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Biochemistry_Lippinco. than nonsmokers with the deficiency.] The deficiency of elastase inhibitor can be treated by weekly augmentation therapy, that is, intravenous administration of AAT. The AAT diffuses from the blood into the lung, where it reaches therapeutic levels in the fluid surrounding the lung epithelial cells.


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Biochemistry_Lippinco. IV. CHAPTER SUMMARY Collagen and elastin are structural fibrous proteins of the extracellular matrix (Fig. 4.15). Collagen contains an abundance of proline, lysine, and glycine, the latter occurring at every third position in the primary structure. It also contains hydroxyproline, hydroxylysine, and glycosylated hydroxylysine, each formed by posttranslational modification. Fibrillar collagen has a long, rigid structure, in which three collagen polypeptide α chains are wound around one another in a rope-like triple helix stabilized by interchain hydrogen bonds. Diseases of fibrillar collagen synthesis affect bones, joints, skin, and blood vessels. Elastin is a connective tissue protein with rubber-like properties in tissues such as the lung. α1-Antitrypsin (AAT), produced primarily by the liver, inhibits elastase-catalyzed degradation of elastin in the alveolar walls. A deficiency of AAT increases elastin degradation and can cause emphysema and, in some cases, cirrhosis of the liver.


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Biochemistry_Lippinco. Choose the ONE best answer. .1. A 30-year-old woman of Northern European ancestry presents with progressive dyspnea (shortness of breath). She denies the use of cigarettes. Family history reveals that her sister also has problems with her lungs. Which one of the following etiologies most likely explains this patient’s pulmonary symptoms? A. Deficiency in dietary vitamin C B. Deficiency of α1-antitrypsin C. Deficiency of prolyl hydroxylase D. Decreased elastase activity E. Increased collagenase activity


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Biochemistry_Lippinco. A. Deficiency in dietary vitamin C B. Deficiency of α1-antitrypsin C. Deficiency of prolyl hydroxylase D. Decreased elastase activity E. Increased collagenase activity Correct answer = B. α1-Antitrypsin (AAT) deficiency is a genetic disorder that can cause pulmonary damage and emphysema even in the absence of cigarette use. A deficiency of AAT permits increased elastase activity to destroy elastin in the alveolar walls. AAT deficiency should be suspected when chronic obstructive pulmonary disease develops in a patient younger than age 45 years who does not have a history of chronic bronchitis or tobacco use or when multiple family members develop obstructive lung disease at an early age. Choices A, C, and E refer to collagen, not elastin.


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Biochemistry_Lippinco. .3. A 7-month-old child “fell over” while crawling and now presents with a swollen leg. Imaging reveals a fracture of a bowed femur, secondary to minor trauma, and thin bones (see x-ray at right). Blue sclerae are also noted. At age 1 month, the infant had multiple fractures in various states of healing (right clavicle, right humerus, and right radius). A careful family history has ruled out nonaccidental trauma (child abuse) as a cause of the bone fractures. Which pairing of a defective (or deficient) molecule and the resulting pathology best fits this clinical description? A. Elastin and emphysema B. Fibrillin and Marfan disease C. Type I collagen and osteogenesis imperfecta D. Type V collagen and Ehlers-Danlos syndrome E. Vitamin C and scurvy


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Biochemistry_Lippinco. A. Elastin and emphysema B. Fibrillin and Marfan disease C. Type I collagen and osteogenesis imperfecta D. Type V collagen and Ehlers-Danlos syndrome E. Vitamin C and scurvy Correct answer = C. The child most likely has osteogenesis imperfecta. Most cases arise from a defect in the genes encoding type I collagen. Bones in affected patients are thin, osteoporotic, often bowed, and extremely prone to fracture. Pulmonary problems are not seen in this child. Individuals with Marfan syndrome have impaired structural integrity of the skeleton, eyes, and cardiovascular system. Defects in type V collagen cause the classic form of Ehlers-Danlos syndrome characterized by skin extensibility and fragility and joint hypermobility. Scurvy caused by vitamin C deficiency is characterized by capillary fragility. .2. What is the differential basis of the liver and lung pathology seen in α1antitrypsin deficiency?


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Biochemistry_Lippinco. .2. What is the differential basis of the liver and lung pathology seen in α1antitrypsin deficiency? With α1-antitrypsin (AAT) deficiency, the cirrhosis that can result is due to polymerization and retention of AAT in the liver, its site of synthesis. The alveolar damage is due to the retention-based deficiency of AAT (a serine protease inhibitor) in the lung such that elastase (a serine protease) is unopposed. .4. How and why is proline hydroxylated in collagen? Proline is hydroxlyated by prolyl hydroxylase, an enzyme of the endoplasmic reticulum that requires oxygen, ferrous iron, and vitamin C. Hydroxylation increases interchain hydrogen bond formation, strengthening the triple helix of collagen. Vitamin C deficiency impairs hydroxylation. For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW


Biochemistry_Lippinco

Biochemistry_Lippinco. For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW Virtually all reactions in the body are mediated by enzymes, which are protein catalysts that increase the rate of reactions without being changed in the overall process. Among the many biologic reactions that are energetically possible, enzymes selectively channel reactants (called substrates) into useful pathways. Thus, enzymes direct all metabolic events. This chapter examines the nature of these catalytic molecules and their mechanisms of action. II. NOMENCLATURE Each enzyme is assigned two names. The first is its short, recommended name, convenient for everyday use. The second is the more complete systematic name, which is used when an enzyme must be identified without ambiguity. A. Recommended name


Biochemistry_Lippinco

Biochemistry_Lippinco. A. Recommended name Most commonly used enzyme names have the suffix “-ase” attached to the substrate of the reaction (for example, glucosidase and urease) or to a description of the action performed (for example, lactate dehydrogenase and adenylyl cyclase). [Note: Some enzymes retain their original trivial names, which give no hint of the associated enzymic reaction, for example, trypsin and pepsin.] B. Systematic name In the systematic naming system, enzymes are divided into six major classes (Fig. 5.1), each with numerous subgroups. For a given enzyme, the suffix ase is attached to a fairly complete description of the chemical reaction catalyzed, including the names of all the substrates, for example, lactate:nicotinamide adenine dinucleotide (NAD+) oxidoreductase. [Note: Each enzyme is also assigned a classification number. Lactate:NAD+ oxidoreductase is 1.1.1.27.] The systematic names are unambiguous and informative but are frequently too cumbersome to be of general use.


Biochemistry_Lippinco

Biochemistry_Lippinco. inorganic phosphate. Potentially confusing enzyme nomenclature includes synthetase (requires ATP), synthase (no ATP required), phosphatase (uses water to remove a phosphate group), phosphorylase (uses inorganic phosphate to break a bond and generate a phosphorylated product), dehydrogenase (NAD+ or flavin adenine dinucleotide [FAD] is an electron acceptor in a redox reaction), oxidase (oxygen is the acceptor, and oxygen atoms are not incorporated into substrate), and oxygenase (one or both oxygen atoms are incorporated). III. PROPERTIES Enzymes are protein catalysts that increase the velocity of a chemical reaction and are not consumed during the reaction. [Note: Some ribonucleic acids (RNA) can catalyze reactions that affect phosphodiester and peptide bonds. RNAs with catalytic activity are called ribozymes (see p. 434) and are much less common than protein catalysts.] A. Active site


Biochemistry_Lippinco

Biochemistry_Lippinco. A. Active site Enzyme molecules contain a special pocket or cleft called the active site. The active site, formed by folding of the protein, contains amino acid side chains that participate in substrate binding and catalysis (Fig. 5.2). The substrate binds the enzyme noncovalently, forming an enzyme–substrate (ES) complex. Binding is thought to cause a conformational change in the enzyme (induced fit model) that allows catalysis. ES is converted to an enzyme–product (EP) complex that subsequently dissociates to enzyme and product. B. Efficiency Enzyme-catalyzed reactions are highly efficient, proceeding from 103 to 108 times faster than uncatalyzed reactions. The number of substrate molecules converted to product per enzyme molecule per second is called the turnover number, or kcat, and typically is 102–104 s−1 . [Note: kcat is the rate constant for the conversion of ES to E + P (see p. 58).] C. Specificity


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Biochemistry_Lippinco. C. Specificity Enzymes are highly specific, interacting with one or a few substrates and catalyzing only one type of chemical reaction. The set of enzymes made in a cell determines which reactions occur in that cell. D. Holoenzymes, apoenzymes, cofactors, and coenzymes


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Biochemistry_Lippinco. D. Holoenzymes, apoenzymes, cofactors, and coenzymes Some enzymes require nonproteins for enzymic activity. The term holoenzyme refers to the active enzyme with its nonprotein component, whereas the enzyme without its nonprotein moiety is termed an apoenzyme and is inactive. If the nonprotein moiety is a metal ion, such as zinc (Zn2+) or iron (Fe2+), it is called a cofactor (see Chapter 29). If it is a small organic molecule, it is termed a coenzyme. Coenzymes that only transiently associate with the enzyme are called cosubstrates. Cosubstrates dissociate from the enzyme in an altered state (NAD+ is an example; see p. 101). If the coenzyme is permanently associated with the enzyme and returned to its original form, it is called a prosthetic group (FAD is an example; see p. 110). Coenzymes commonly are derived from vitamins. For example, NAD+ contains niacin, and FAD contains riboflavin (see Chapter 28). E. Regulation


Biochemistry_Lippinco

Biochemistry_Lippinco. E. Regulation Enzyme activity can be regulated, that is, increased or decreased, so that the rate of product formation responds to cellular need. F. Location within the cell Many enzymes are localized in specific organelles within the cell (Fig. 5.3). Such compartmentalization serves to isolate the reaction substrate or product from other competing reactions. This provides a favorable environment for the reaction and organizes the thousands of enzymes present in the cell into purposeful pathways. IV. MECHANISM OF ENZYME ACTION The mechanism of enzyme action can be viewed from two different perspectives. The first treats catalysis in terms of energy changes that occur during the reaction. That is, enzymes provide an alternate, energetically favorable reaction pathway different from the uncatalyzed reaction. The second perspective describes how the active site chemically facilitates catalysis. A. Energy changes occurring during the reaction


Biochemistry_Lippinco

Biochemistry_Lippinco. A. Energy changes occurring during the reaction Virtually all chemical reactions have an energy barrier separating the reactants and the products. This barrier, called the activation energy (Ea), is the energy difference between that of the reactants and a high-energy intermediate, the transition state (T*), which is formed during the conversion of reactant to product. Figure 5.4 shows the changes in energy during the conversion of a molecule of reactant A to product B as it proceeds through the transition state. 1. Activation energy: The peak of energy in Figure 5.4 is the difference in free energy between the reactant and T*, in which the high-energy, short-lived intermediate is formed during the conversion of reactant to product. Because of the high Ea, the rates of uncatalyzed chemical reactions are often slow. 2.


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Biochemistry_Lippinco. 2. Rate of reaction: For molecules to react, they must contain sufficient energy to overcome the energy barrier of the transition state. In the absence of an enzyme, only a small proportion of a population of molecules may possess enough energy to achieve the transition state between reactant and product. The rate of reaction is determined by the number of such energized molecules. In general, the lower the Ea, the more molecules have sufficient energy to pass through the transition state and, therefore, the faster the rate of the reaction. 3. Alternate reaction pathway: An enzyme allows a reaction to proceed rapidly under conditions prevailing in the cell by providing an alternate reaction pathway with a lower Ea (see Fig. 5.4). The enzyme does not change the free energies of the reactants (substrates) or products and, therefore, does not change the equilibrium of the reaction (see p. 70). It does, however, accelerate the rate by which equilibrium is reached.


Biochemistry_Lippinco

Biochemistry_Lippinco. B. Active site chemistry The active site is not a passive receptacle for binding the substrate but, rather, is a complex molecular machine employing a diversity of chemical mechanisms to facilitate the conversion of substrate to product. A number of factors are responsible for the catalytic efficiency of enzymes, including the following examples. 1. Transition-state stabilization: The active site often acts as a flexible molecular template that binds the substrate and initiates its conversion to the transition state, a structure in which the bonds are not like those in the substrate or the product (see T* at the top of the curve in Fig. 5.4). By stabilizing the transition state, the enzyme greatly increases the concentration of the reactive intermediate that can be converted to product and, thus, accelerates the reaction. [Note: The transition state cannot be isolated.] 2.


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Biochemistry_Lippinco. Catalysis: The active site can provide catalytic groups that enhance the probability that the transition state is formed. In some enzymes, these groups can participate in general acid–base catalysis in which amino acid residues provide or accept protons. In other enzymes, catalysis may involve the transient formation of a covalent ES complex. [Note: The mechanism of action of chymotrypsin, an enzyme of protein digestion in the intestine, includes general base, general acid, and covalent catalysis. A histidine at the active site of the enzyme gains (general base) and loses (general acid) protons, mediated by the pK of histidine in proteins being close to physiologic pH. Serine at the active site forms a transient covalent bond with the substrate.] 3.


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Biochemistry_Lippinco. Transition-state visualization: The enzyme-catalyzed conversion of substrate to product can be visualized as being similar to removing a sweater from an uncooperative infant (Fig. 5.5). The process has a high Ea because the only reasonable strategy for removing the garment (short of ripping it off) requires that the random flailing of the baby results in both arms being fully extended over the head, an unlikely posture. However, we can envision a parent acting as an enzyme, first coming in contact with the baby (forming ES) and then guiding the baby’s arms into an extended, vertical position, analogous to the transition state. This posture (conformation) of the baby facilitates the removal of the sweater, forming the disrobed baby, which here represents product. [Note: The substrate bound to the enzyme (ES) is at a slightly lower energy than unbound substrate (S) and explains the small dip in the curve at ES.] V. FACTORS AFFECTING REACTION VELOCITY


Biochemistry_Lippinco

Biochemistry_Lippinco. V. FACTORS AFFECTING REACTION VELOCITY Enzymes can be isolated from cells and their properties studied in a test tube (that is, in vitro). Different enzymes show different responses to changes in substrate concentration, temperature, and pH. This section describes factors that influence the reaction velocity of enzymes. Enzymic responses to these factors give us valuable clues as to how enzymes function in living cells (that is, in vivo). A. Substrate concentration 1. Maximal velocity: The rate or velocity of a reaction (v) is the number of substrate molecules converted to product per unit time. Velocity is usually expressed as µmol of product formed per minute. The rate of an enzyme-catalyzed reaction increases with substrate concentration until a maximal velocity (Vmax) is reached (Fig. 5.6). The leveling off of the reaction rate at high substrate concentrations reflects the saturation with substrate of all available binding sites on the enzyme molecules present. 2.


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Biochemistry_Lippinco. 2. Shape of the enzyme kinetics curve: Most enzymes show Michaelis-Menten kinetics (see p. 58), in which the plot of initial reaction velocity (vo) against substrate concentration is hyperbolic (similar in shape to that of the oxygen-dissociation curve of myoglobin; see p. 29). In contrast, allosteric enzymes do not follow Michaelis-Menten kinetics and show a sigmoidal curve (see Fig. 5.6) that is similar in shape to the oxygen-dissociation curve of hemoglobin (see p. 29). B. Temperature 1. Velocity increase with temperature: The reaction velocity increases with temperature until a peak velocity is reached (Fig. 5.7). This increase is the result of the increased number of substrate molecules having sufficient energy to pass over the energy barrier and form the products of the reaction. 2.


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Biochemistry_Lippinco. 2. Velocity decrease with higher temperature: Further elevation of the temperature causes a decrease in reaction velocity as a result of temperature-induced denaturation of the enzyme (see Fig. 5.7). The optimum temperature for most human enzymes is between 35°C and 40°C. Human enzymes start to denature (see p. 20) at temperatures above 40°C, but thermophilic bacteria found in hot springs have optimum temperatures of 70°C. C. pH 1. pH effect on active site ionization: The concentration of protons ([H+]) affects reaction velocity in several ways. First, the catalytic process usually requires that the enzyme and substrate have specific chemical groups in either an ionized or unionized state in order to interact. For example, catalytic activity may require that an amino group of the enzyme be in the protonated form (−NH3+). Because this group is deprotonated at alkaline pH, the rate of the reaction declines. 2.


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Biochemistry_Lippinco. 2. pH effect on enzyme denaturation: Extremes of pH can also lead to denaturation of the enzyme, because the structure of the catalytically active protein molecule depends on the ionic character of the amino acid side chains. 3. Variable pH optimum: The pH at which maximal enzyme activity is achieved is different for different enzymes and often reflects the [H+] at which the enzyme functions in the body. For example, pepsin, a digestive enzyme in the stomach, is maximally active at pH 2, whereas other enzymes, designed to work at neutral pH, are denatured by such an acidic environment (Fig. 5.8). VI. MICHAELIS-MENTEN KINETICS


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Biochemistry_Lippinco. VI. MICHAELIS-MENTEN KINETICS Leonor Michaelis and Maude Menten proposed a simple model that accounts for most of the features of many enzyme-catalyzed reactions. In this model, the enzyme reversibly combines with its substrate to form an ES complex that subsequently yields product, regenerating the free enzyme. The reaction model, involving one substrate molecule, is represented below: where S is the substrate. E is the enzyme. ES is the enzyme–substrate complex. P is the product. k1, k−1, and k2 (or, kcat) are rate constants. A. Michaelis-Menten equation The Michaelis-Menten equation describes how reaction velocity varies with substrate concentration: The following assumptions are made in deriving the Michaelis-Menten rate equation. 1. Enzyme and substrate relative concentrations: The substrate concentration ([S]) is much greater than the concentration of enzyme so that the percentage of total substrate bound by the enzyme at any one time is small. 2.


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Biochemistry_Lippinco. 2. Steady-state assumption: The concentration of the ES complex does not change with time (the steady-state assumption), that is, the rate of formation of ES is equal to that of the breakdown of ES (to E + S and to E + P). In general, an intermediate in a series of reactions is said to be in steady state when its rate of synthesis is equal to its rate of degradation. 3. Initial velocity: Initial reaction velocities (vo) are used in the analysis of enzyme reactions. This means that the rate of the reaction is measured as soon as enzyme and substrate are mixed. At that time, the concentration of product is very small, and therefore, the rate of the back reaction from product to substrate can be ignored.


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Biochemistry_Lippinco. B. Important conclusions 1. Km characteristics: Km, the Michaelis constant, is characteristic of an enzyme and its particular substrate and reflects the affinity of the enzyme for that substrate. Km is numerically equal to the substrate concentration at which the reaction velocity is equal to one half Vmax. Km does not vary with enzyme concentration. a. Small Km: A numerically small (low) Km reflects a high affinity of the enzyme for substrate, because a low concentration of substrate is needed to half-saturate the enzyme—that is, to reach a velocity that is one half Vmax (Fig. 5.9). b. Large Km: A numerically large (high) Km reflects a low affinity of enzyme for substrate because a high concentration of substrate is needed to half-saturate the enzyme. 2.


Biochemistry_Lippinco

Biochemistry_Lippinco. b. Large Km: A numerically large (high) Km reflects a low affinity of enzyme for substrate because a high concentration of substrate is needed to half-saturate the enzyme. 2. Velocity relationship to enzyme concentration: The rate of the reaction is directly proportional to the enzyme concentration because [S] is not limiting. For example, if the enzyme concentration is halved, the initial rates of the reaction (vo) and that of Vmax are reduced to half that of the original. 3. Reaction order: When [S] is much less (<<) than Km, the velocity of the reaction is approximately proportional to the substrate concentration (Fig. 5.10). The rate of reaction is then said to be first order with respect to substrate. When [S] is much greater (>>) than Km, the velocity is constant and equal to Vmax. The rate of reaction is then independent of substrate concentration (the enzyme is saturated with substrate) and is said to be zero order with respect to substrate concentration (see Fig. 5.10).


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Biochemistry_Lippinco. D. Lineweaver-Burk plot When vo is plotted against [S], it is not always possible to determine when Vmax has been achieved because of the gradual upward slope of the hyperbolic curve at high substrate concentrations. However, if 1/vo is plotted versus 1/[S], a straight line is obtained (Fig. 5.11). This plot, the Lineweaver-Burk plot (also called a double-reciprocal plot) can be used to calculate Km and Vmax as well as to determine the mechanism of action of enzyme inhibitors. The equation describing the Lineweaver-Burk plot is: where the intercept on the x axis is equal to − 1/Km, and the intercept on the y axis is equal to 1/Vmax. [Note: The slope = Km/Vmax.] VII. ENZYME INHIBITION


Biochemistry_Lippinco

Biochemistry_Lippinco. VII. ENZYME INHIBITION Any substance that can decrease the velocity of an enzyme-catalyzed reaction is called an inhibitor. Inhibitors can be reversible or irreversible. Irreversible inhibitors bind to enzymes through covalent bonds. Lead, for example, forms covalent bonds with the sulfhydryl side chain of cysteine in proteins. Ferrochelatase, an enzyme involved in heme synthesis (see p. 279), is irreversibly inhibited by lead. [Note: An important group of irreversible inhibitors are the mechanism-based inhibitors that are converted by the enzyme itself to a form that covalently links to the enzyme, thereby inhibiting it. They also are referred to as “suicide” inhibitors.] Reversible inhibitors bind to enzymes through noncovalent bonds and, thus, dilution of the enzyme–inhibitor complex results in dissociation of the reversibly bound inhibitor and recovery of enzyme activity. The two most commonly encountered types of reversible inhibition are competitive and noncompetitive.


Biochemistry_Lippinco

Biochemistry_Lippinco. A. Competitive inhibition This type of inhibition occurs when the inhibitor binds reversibly to the same site that the substrate would normally occupy and, therefore, competes with the substrate for that site. 1. Effect on Vmax: The effect of a competitive inhibitor is reversed by increasing the concentration of substrate. At a sufficiently high [S], the 2. Effect on Km: A competitive inhibitor increases the apparent Km for a given substrate. This means that, in the presence of a competitive inhibitor, more substrate is needed to achieve one half Vmax. 3. Effect on the Lineweaver-Burk plot: Competitive inhibition shows a characteristic Lineweaver-Burk plot in which the plots of the inhibited and uninhibited reactions intersect on the y axis at 1/Vmax (Vmax is reaction velocity reaches the Vmax observed in the absence of inhibitor, that is, Vmax is unchanged (Fig. 5.12).


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Biochemistry_Lippinco. unchanged). The inhibited and uninhibited reactions show different x-axis intercepts, indicating that the apparent Km is increased in the presence of the competitive inhibitor because − 1/Km moves closer to zero from a negative value (see Fig. 5.12). [Note: An important group of competitive inhibitors are the transition state analogs, stable molecules that approximate the structure of the transition state, and, therefore, bind the enzyme more tightly than does the substrate.] 4. Statin drugs as examples of competitive inhibitors: This group of antihyperlipidemic agents competitively inhibits the rate-limiting (slowest) step in cholesterol biosynthesis. This reaction is catalyzed by hydroxymethylglutaryl coenzyme A reductase (HMG CoA reductase; see p. 221). Statins, such as atorvastatin (Lipitor) and pravastatin (Pravachol), are structural analogs of the natural substrate for this enzyme and compete effectively to inhibit HMG CoA reductase. By doing so, they inhibit de novo cholesterol


Biochemistry_Lippinco

Biochemistry_Lippinco. and pravastatin (Pravachol), are structural analogs of the natural substrate for this enzyme and compete effectively to inhibit HMG CoA reductase. By doing so, they inhibit de novo cholesterol synthesis, thereby lowering plasma cholesterol levels (Fig. 5.13).


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Biochemistry_Lippinco. B. Noncompetitive inhibition This type of inhibition is recognized by its characteristic effect on Vmax (Fig. 5.14). Noncompetitive inhibition occurs when the inhibitor and substrate bind at different sites on the enzyme. The noncompetitive inhibitor can bind either free enzyme or the enzyme–substrate complex, thereby preventing the reaction from occurring (Fig. 5.15). 1. Effect on Vmax: Noncompetitive inhibition cannot be overcome by increasing the concentration of substrate. Therefore, noncompetitive inhibitors decrease the apparent Vmax of the reaction. 2. Effect on Km: Noncompetitive inhibitors do not interfere with the binding of substrate to enzyme. Therefore, the enzyme shows the same Km in the presence or absence of the noncompetitive inhibitor, that is, Km is unchanged. 3.


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Biochemistry_Lippinco. 3. Effect on Lineweaver-Burk plot: Noncompetitive inhibition is readily differentiated from competitive inhibition by plotting 1/vo versus 1/[S] and noting that the apparent Vmax decreases in the presence of a noncompetitive inhibitor, whereas Km is unchanged (see Fig. 5.14). [Note: Oxypurinol, a metabolite of the prodrug allopurinol, is a noncompetitive inhibitor of xanthine oxidase, an enzyme of purine degradation (see p. 301).] C. Enzyme inhibitors as drugs


Biochemistry_Lippinco

Biochemistry_Lippinco. C. Enzyme inhibitors as drugs At least half of the ten most commonly prescribed drugs in the United States act as enzyme inhibitors. For example, the widely prescribed βlactam antibiotics, such as penicillin and amoxicillin, act by inhibiting enzymes involved in bacterial cell wall synthesis. Drugs may also act by inhibiting extracellular reactions. This is illustrated by angiotensinconverting enzyme (ACE) inhibitors. They lower blood pressure by blocking plasma ACE that cleaves angiotensin I to form the potent vasoconstrictor, angiotensin II. These drugs, which include captopril, enalapril, and lisinopril, cause vasodilation and, therefore, a reduction in blood pressure. Aspirin, a nonprescription drug, irreversibly inhibits prostaglandin and thromboxane synthesis by inhibiting cyclooxygenase (see p. 214). VIII. ENZYME REGULATION


Biochemistry_Lippinco

Biochemistry_Lippinco. VIII. ENZYME REGULATION The regulation of the reaction velocity of enzymes is essential if an organism is to coordinate its numerous metabolic processes. The rates of most enzymes are responsive to changes in substrate concentration, because the intracellular level of many substrates is in the range of the Km. Thus, an increase in substrate concentration prompts an increase in reaction rate, which tends to return the concentration of substrate toward normal. In addition, some enzymes with specialized regulatory functions respond to allosteric effectors and/or covalent modification or they show altered rates of enzyme synthesis (or degradation) when physiologic conditions are changed. A. Allosteric enzymes


Biochemistry_Lippinco

Biochemistry_Lippinco. A. Allosteric enzymes Allosteric enzymes are regulated by molecules called effectors that bind noncovalently at a site other than the active site. These enzymes are almost always composed of multiple subunits, and the regulatory (allosteric) site that binds the effector is distinct from the substrate-binding site and may be located on a subunit that is not itself catalytic. Effectors that inhibit enzyme activity are termed negative effectors, whereas those that increase enzyme activity are called positive effectors. Positive and negative effectors can affect the affinity of the enzyme for its substrate (K0.5), modify the maximal catalytic activity of the enzyme (Vmax), or both (Fig. 5.16). [Note: Allosteric enzymes frequently catalyze the committed step, often the rate-limiting step, early in a pathway.] 1.


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Biochemistry_Lippinco. Homotropic effectors: When the substrate itself serves as an effector, the effect is said to be homotropic. Most often, an allosteric substrate functions as a positive effector. In such a case, the presence of a substrate molecule at one site on the enzyme enhances the catalytic properties of the other substrate-binding sites. That is, their binding sites exhibit cooperativity. These enzymes show a sigmoidal curve when vo is plotted against substrate concentration, as shown in Figure 5.16. This contrasts with the hyperbolic curve characteristic of enzymes following Michaelis-Menten kinetics, as previously discussed. [Note: The concept of cooperativity of substrate binding is analogous to the binding of oxygen to hemoglobin (see p. 29).] 2.


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Biochemistry_Lippinco. Heterotropic effectors: The effector may be different from the substrate, in which case the effect is said to be heterotropic. For example, consider the feedback inhibition shown in Figure 5.17. The enzyme that converts D to E has an allosteric site that binds the end product, G. If the concentration of G increases (for example, because it is not used as rapidly as it is synthesized), the first irreversible step unique to the pathway is typically inhibited. Feedback inhibition provides the cell with appropriate amounts of a product it needs by regulating the flow of substrate molecules through the pathway that synthesizes that product. Heterotropic effectors are commonly encountered. For example, the glycolytic enzyme phosphofructokinase-1 is allosterically inhibited by citrate, which is not a substrate for the enzyme (see p. 99). Figure5.17Feedbackinhibitionofametabolicpathway. B. Covalent modification


Biochemistry_Lippinco

Biochemistry_Lippinco. Figure5.17Feedbackinhibitionofametabolicpathway. B. Covalent modification Many enzymes are regulated by covalent modification, most often by the addition or removal of phosphate groups from specific serine, threonine, or tyrosine residues of the enzyme. Protein phosphorylation is recognized as one of the primary ways in which cellular processes are regulated. 1. Phosphorylation and dephosphorylation: Phosphorylation reactions are catalyzed by a family of enzymes called protein kinases that use ATP as the phosphate donor. Phosphate groups are cleaved from phosphorylated enzymes by the action of phosphoprotein phosphatases (Fig. 5.18). 2.


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Biochemistry_Lippinco. 2. Enzyme response to phosphorylation: Depending on the specific enzyme, the phosphorylated form may be more or less active than the unphosphorylated enzyme. For example, hormone-mediated phosphorylation of glycogen phosphorylase (an enzyme that degrades glycogen) increases activity, whereas phosphorylation of glycogen synthase (an enzyme that synthesizes glycogen) decreases activity (see p. 132). C. Enzyme synthesis


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Biochemistry_Lippinco. The regulatory mechanisms described above modify the activity of existing enzyme molecules. However, cells can also regulate the amount of enzyme present by altering the rate of enzyme degradation or, more typically, the rate of enzyme synthesis. The increase (induction) or decrease (repression) of enzyme synthesis leads to an alteration in the total population of active sites. Enzymes subject to regulation of synthesis are often those that are needed at only one stage of development or under selected physiologic conditions. For example, elevated levels of insulin as a result of high blood glucose levels cause an increase in the synthesis of key enzymes involved in glucose metabolism (see p. 105). In contrast, enzymes that are in constant use are usually not regulated by altering the rate of enzyme synthesis. Alterations in enzyme levels as a result of induction or repression of protein synthesis are slow (hours to days), compared with allosterically or covalently regulated changes in


Biochemistry_Lippinco

Biochemistry_Lippinco. of enzyme synthesis. Alterations in enzyme levels as a result of induction or repression of protein synthesis are slow (hours to days), compared with allosterically or covalently regulated changes in enzyme activity, which occur in seconds to minutes. Figure 5.19 summarizes the common ways that enzyme activity is regulated.


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Biochemistry_Lippinco. IX. Enzymes in Clinical Diagnosis Plasma enzymes can be classified into two major groups. First, a relatively small group of enzymes are actively secreted into the blood by certain cell types. For example, the liver secretes zymogens (inactive precursors) of the enzymes involved in blood coagulation. Second, a large number of enzyme species are released from cells during normal cell turnover. These enzymes almost always function intracellularly and have no physiologic use in the plasma. In healthy individuals, the levels of these enzymes are fairly constant and represent a steady state in which the rate of release from damaged cells into the plasma is balanced by an equal rate of removal from the plasma. Increased plasma levels of these enzymes may indicate tissue damage (Fig. 5.20). (B) cells.


Biochemistry_Lippinco

Biochemistry_Lippinco. (B) cells. Plasma is the fluid, noncellular part of blood. Laboratory assays of enzyme activity most often use serum, which is obtained by centrifugation of whole blood after it has been allowed to coagulate. Plasma is a physiologic fluid, whereas serum is prepared in the laboratory. A. Plasma enzyme levels in disease states Many diseases that cause tissue damage result in an increased release of intracellular enzymes into the plasma. The activities of many of these enzymes are routinely determined for diagnostic purposes in diseases of the heart, liver, skeletal muscle, and other tissues. The level of specific enzyme activity in the plasma frequently correlates with the extent of tissue damage. Therefore, determining the degree of elevation of a particular enzyme activity in the plasma is often useful in evaluating the prognosis for the patient. B. Plasma enzymes as diagnostic tools


Biochemistry_Lippinco

Biochemistry_Lippinco. B. Plasma enzymes as diagnostic tools Some enzymes show relatively high activity in only one or a few tissues. Therefore, the presence of increased levels of these enzymes in plasma reflects damage to the corresponding tissue. For example, the enzyme alanine aminotransferase (ALT; see p. 251) is abundant in the liver. The appearance of elevated levels of ALT in plasma signals possible damage to hepatic tissue. [Note: Measurement of ALT is part of the liver function test panel.] Increases in plasma levels of enzymes with a wide tissue distribution provide a less specific indication of the site of cellular injury and limits their diagnostic value. C. Isoenzymes and heart disease


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Biochemistry_Lippinco. C. Isoenzymes and heart disease Isoenzymes (also called isozymes) are enzymes that catalyze the same reaction. However, they do not necessarily have the same physical properties because of genetically determined differences in amino acid sequence. For this reason, isoenzymes may contain different numbers of charged amino acids, which allows electrophoresis (the movement of charged particles in an electric field) to separate them (Fig. 5.21). Different organs commonly contain characteristic proportions of different isoenzymes. The pattern of isoenzymes found in the plasma may, therefore, serve as a means of identifying the site of tissue damage. For example, the plasma levels of creatine kinase (CK) are commonly determined in the diagnosis of myocardial infarction (MI). They are particularly useful when the electrocardiogram (ECG) is difficult to interpret such as when there have been previous episodes of heart disease. 1.


Biochemistry_Lippinco

Biochemistry_Lippinco. 1. Isoenzyme quaternary structure: Many isoenzymes contain different subunits in various combinations. For example, CK occurs as three isoenzymes. Each isoenzyme is a dimer composed of two polypeptides (called B and M subunits) associated in one of three combinations: CK1 = BB, CK2 = MB, and CK3 = MM. Each CK isoenzyme shows a characteristic electrophoretic mobility (see Fig. 5.21). [Note: Virtually all CK in the brain is the BB isoform, whereas it is MM in skeletal muscle. In cardiac muscle, about one third is MB with the rest as MM.] 2.


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Biochemistry_Lippinco. Diagnosis of myocardial infarction: Measurement of blood levels of proteins with cardiac specificity (biomarkers) is used in the diagnosis of MI. Myocardial muscle is the only tissue that contains >5% of the total CK activity as the CK2 (MB) isoenzyme. Appearance of this hybrid isoenzyme in plasma is virtually specific for infarction of the myocardium. Following an acute MI, CK2 appears in plasma within 4–8 hours following onset of chest pain, reaches a peak of activity at ~24 hours, and returns to baseline after 48–72 hours (Fig. 5.22). Troponins T (TnT) and I (TnI) are regulatory proteins involved in muscle contractility. Cardiac-specific isoforms (cTn) are released into the plasma in response to cardiac damage. They are highly sensitive and specific for damage to cardiac tissue. cTn appear in plasma within 4–6 hours after an MI, peak in 24–36 hours, and remain elevated for 3–10 days. Elevated cTn, in combination with the clinical presentation and characteristic changes in the ECG,


Biochemistry_Lippinco

Biochemistry_Lippinco. in plasma within 4–6 hours after an MI, peak in 24–36 hours, and remain elevated for 3–10 days. Elevated cTn, in combination with the clinical presentation and characteristic changes in the ECG, are currently considered the “gold standard” in the diagnosis of an MI.


Biochemistry_Lippinco

X. CHAPTER SUMMARY

Biochemistry_Lippinco. X. CHAPTER SUMMARY


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Biochemistry_Lippinco. Enzymes are protein catalysts that increase the velocity of a chemical reaction by lowering the energy of the transition state (Fig. 5.23). They are not consumed during the reaction. Enzyme molecules contain a special cleft called the active site, which contains amino acid side chains that participate in substrate binding and catalysis. The active site binds the substrate, forming an enzyme–substrate (ES) complex. Binding is thought to cause a conformational change in the enzyme (induced fit) that allows catalysis. ES is converted to enzyme and product. An enzyme allows a reaction to proceed rapidly under conditions prevailing in the cell by providing an alternate reaction pathway with a lower activation energy (Ea). Because the enzyme does not change the free energies of the reactants or products, it does not change the equilibrium of the reaction. Most enzymes show Michaelis-Menten kinetics, and a plot of the initial reaction velocity (vo) against substrate concentration ([S]) has a


Biochemistry_Lippinco

Biochemistry_Lippinco. products, it does not change the equilibrium of the reaction. Most enzymes show Michaelis-Menten kinetics, and a plot of the initial reaction velocity (vo) against substrate concentration ([S]) has a hyperbolic shape similar to the oxygen-dissociation curve of myoglobin. A Lineweaver-Burk plot of 1/v and 1/[S] allows determination of Vmax (maximal velocity) and Km (Michaelis constant, which reflects affinity for substrate). Any substance that can decrease the velocity of an enzyme-catalyzed reaction is called an inhibitor. The two most common types of reversible inhibition are competitive (which increases the apparent Km) and noncompetitive (which decreases the apparent Vmax). In contrast, the multisubunit allosteric enzymes show a sigmoidal curve similar in shape to the oxygen-dissociation curve of hemoglobin. They typically catalyze the committed step of a pathway. Allosteric enzymes are regulated by molecules called effectors that bind noncovalently at a site other than the active


Biochemistry_Lippinco

Biochemistry_Lippinco. curve of hemoglobin. They typically catalyze the committed step of a pathway. Allosteric enzymes are regulated by molecules called effectors that bind noncovalently at a site other than the active site. Effectors can be either positive (increase enzyme activity) or negative (decrease enzyme activity). An allosteric effector can alter the affinity of the enzyme for its substrate (K0.5), the maximal catalytic activity of the enzyme (Vmax), or both. Enzymes can also be regulated by covalent modification and by changes in the rate of synthesis or degradation.


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Biochemistry_Lippinco. Enzymes have diagnostic and therapeutic value in medicine. Choose the ONE best answer. .1. In cases of ethylene glycol poisoning and its characteristic metabolic acidosis, treatment involves correction of the acidosis, removal of any remaining ethylene glycol, and administration of an inhibitor of alcohol dehydrogenase (ADH), the enzyme that oxidizes ethylene glycol to the organic acids that cause the acidosis. Ethanol (grain alcohol) frequently is the inhibitor given to treat ethylene glycol poisoning. Results of experiments using ADH with and without ethanol are shown to the right. Based on these data, what type of inhibition is caused by the ethanol? A. Competitive B. Feedback C. Irreversible D. Noncompetitive


Biochemistry_Lippinco

Biochemistry_Lippinco. A. Competitive B. Feedback C. Irreversible D. Noncompetitive Correct answer = A. A competitive inhibitor increases the apparent Km for a given substrate. This means that, in the presence of a competitive inhibitor, more substrate is needed to achieve one half Vmax. The effect of a competitive inhibitor is reversed by increasing substrate concentration ([S]). At a sufficiently high [S], the reaction velocity reaches the Vmax observed in the absence of inhibitor. .2. Alcohol dehydrogenase (ADH) requires oxidized nicotinamide adenine dinucleotide (NAD+) for catalytic activity. In the reaction catalyzed by ADH, an alcohol is oxidized to an aldehyde as NAD+ is reduced to NADH and dissociates from the enzyme. The NAD+ is functioning as a/an: A. apoenzyme. B. coenzyme–cosubstrate. C. coenzyme–prosthetic group. D. cofactor. E. heterotropic effector.


Biochemistry_Lippinco

Biochemistry_Lippinco. A. apoenzyme. B. coenzyme–cosubstrate. C. coenzyme–prosthetic group. D. cofactor. E. heterotropic effector. Correct answer = B. A Coenzymes–cosubstrates are small organic molecules that associate transiently with an enzyme and leave the enzyme in a changed form. Coenzyme–prosthetic groups are small organic molecules that associate permanently with an enzyme and are returned to their original form on the enzyme. Cofactors are metal ions. Heterotropic effectors are not substrates. For Questions 5.3 and 5.4, use the graph below that shows the changes in free energy when a reactant is converted to a product in the presence and absence of an enzyme. Select the letter that best represents: .3. the activation energy of the catalyzed forward reaction. .4. the free energy of the reaction.


Biochemistry_Lippinco

Biochemistry_Lippinco. Correct answers = B; D. Enzymes (protein catalysts) provide an alternate reaction pathway with a lower activation energy. However, they do not change the free energy of the reactant or product. A is the activation energy of the uncatalyzed reaction. C is the activation energy of the catalyzed reverse reaction. For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW


Biochemistry_Lippinco

Biochemistry_Lippinco. For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW Bioenergetics describes the transfer and utilization of energy in biologic systems. It concerns the initial and final energy states of the reaction components, not the reaction mechanism or how much time it takes for the chemical change to occur. Bioenergetics makes use of a few basic ideas from the field of thermodynamics, particularly the concept of free energy. Because changes in free energy provide a measure of the energetic feasibility of a chemical reaction, they allow prediction of whether a reaction or process can take place. In short, bioenergetics predicts if a process is possible, whereas kinetics measures the reaction rate (see p. 54). II. FREE ENERGY


Biochemistry_Lippinco

Biochemistry_Lippinco. II. FREE ENERGY The direction and extent to which a chemical reaction proceeds are determined by the degree to which two factors change during the reaction. These are enthalpy (∆H, a measure of the change [∆] in heat content of the reactants and products) and entropy (∆S, a measure of the change in randomness or disorder of the reactants and products), as shown in Figure 6.1. Neither of these thermodynamic quantities by itself is sufficient to determine whether a chemical reaction will proceed spontaneously in the direction it is written. However, when combined mathematically (see Fig. 6.1), enthalpy and entropy can be used to define a third quantity, free energy (G), which predicts the direction in which a reaction will spontaneously proceed. III. FREE ENERGY CHANGE


Biochemistry_Lippinco

Biochemistry_Lippinco. III. FREE ENERGY CHANGE The change in free energy is represented in two ways, ∆G and ∆G0. The first, ∆G (without the superscript “0”), represents the change in free energy and, thus, the direction of a reaction at any specified concentration of products and reactants. ∆G, then, is a variable. This contrasts with the standard free energy change, ∆G0 (with the superscript “0”), which is the energy change when reactants and products are at a concentration of 1 mol/l. [Note: The concentration of protons (H+) is assumed to be 10−7 mol/l (that is, pH = 7). This may be shown by a prime sign (ʹ ), for example, ∆G0ʹ.] Although ∆G0, a constant, represents energy changes at these nonphysiologic concentrations of reactants and products, it is nonetheless useful in comparing the energy changes of different reactions. Furthermore, ∆G0 can readily be determined from measurement of the equilibrium constant (see p. 71). [Note: This section outlines the uses of ∆G, and ∆G0 is described in D. below.]


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Biochemistry_Lippinco. A. ∆G and reaction direction The sign of ∆G can be used to predict the direction of a reaction at constant temperature and pressure. Consider the reaction: 1. Negative ∆G: If ∆G is negative, then there is a net loss of energy, and the reaction goes spontaneously as written (that is, A is converted into B) as shown in Figure 6.2A. The reaction is said to be exergonic. 2. Positive ∆G: If ∆G is positive, then there is a net gain of energy, and the reaction does not go spontaneously from B to A (Fig. 6.2B). Energy must be added to the system to make the reaction go from B to A. The reaction is said to be endergonic. 3. Zero ∆G: If ∆G = 0, then the reaction is in equilibrium. [Note: When a reaction is proceeding spontaneously (that is, ∆G is negative), the reaction continues until ∆G reaches zero and equilibrium is established.] B. ∆G of the forward and back reactions


Biochemistry_Lippinco

Biochemistry_Lippinco. B. ∆G of the forward and back reactions The free energy of the forward reaction (A → B) is equal in magnitude but opposite in sign to that of the back reaction (B → A). For example, if ∆G of the forward reaction is −5 kcal/mol, then that of the back reaction is +5 kcal/mol. [Note: ∆G can also be expressed in kilojoules per mole or kJ/mol (1 kcal = 4.2 kJ).] C. ∆G and reactant and product concentrations The ∆G of the reaction A → B depends on the concentration of the reactant and product. At constant temperature and pressure, the following relationship can be derived: where ∆G0 is the standard free energy change (see D. below) R is the gas constant (1.987 cal/mol K) T is the absolute temperature (K) [A] and [B] are the actual concentrations of the reactant and product ln represents the natural logarithm.


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Biochemistry_Lippinco. A reaction with a positive ∆G0 can proceed in the forward direction if the ratio of products to reactants ([B]/[A]) is sufficiently small (that is, the ratio of reactants to products is large) to make ∆G negative. For example, consider the reaction: D. Standard free energy change The standard free energy change, ∆G0, is so called because it is equal to the free energy change, ∆G, under standard conditions (that is, when reactants and products are at 1 mol/l concentrations; Fig. 6.3B). Under these conditions, the natural logarithm of the ratio of products to reactants is zero (ln1 = 0), and, therefore, the equation shown at the bottom of the previous page becomes: 1.


Biochemistry_Lippinco

Biochemistry_Lippinco. ∆G0 and reaction direction: Under standard conditions, ∆G0 can be used to predict the direction a reaction proceeds because, under these conditions, ∆G0 is equal to ∆G. However, ∆G0 cannot predict the direction of a reaction under physiologic conditions because it is composed solely of constants (R, T, and Keq [see 2. below]) and is not, therefore, altered by changes in product or substrate concentrations. 2.


Biochemistry_Lippinco

Biochemistry_Lippinco. 2. Relationship between ∆G0 and Keq: In a reaction A ⇄ B, a point of equilibrium is reached at which no further net chemical change takes place (that is, when A is being converted to B as fast as B is being converted to A). In this state, the ratio of [B] to [A] is constant, regardless of the actual concentrations of the two compounds: where Keq is the equilibrium constant, and [A]eq and [B]eq are the concentrations of A and B at equilibrium. If the reaction A ⇄ B is allowed to go to equilibrium at constant temperature and pressure, then, at equilibrium, the overall ∆G is zero (Fig. 6.3C). Therefore, where the actual concentrations of A and B are equal to the equilibrium concentrations of reactant and product ([A]eq and [B]eq), and their ratio is equal to the Keq. Thus, This equation allows some simple predictions: 3. ∆G0s of two consecutive reactions: The ∆G0s are additive in any sequence of consecutive reactions, as are the ∆Gs. For example: 4.


Biochemistry_Lippinco

Biochemistry_Lippinco. This equation allows some simple predictions: 3. ∆G0s of two consecutive reactions: The ∆G0s are additive in any sequence of consecutive reactions, as are the ∆Gs. For example: 4. ∆Gs of a pathway: The additive property of ∆G is very important in biochemical pathways through which substrates (reactants) must pass in a particular direction (for example, A → B → C → D → …). As long as the sum of the ∆Gs of the individual reactions is negative, the pathway can proceed as written, even if some of the individual reactions of the pathway have a positive ∆G. However, the actual rates of the reactions depend on the lowering of activation energies (Ea) by the enzymes that catalyze the reactions (see p. 55). IV. ATP: AN ENERGY CARRIER


Biochemistry_Lippinco

Biochemistry_Lippinco. IV. ATP: AN ENERGY CARRIER Reactions or processes that have a large positive ∆G, such as moving ions against a concentration gradient across a cell membrane, are made possible by coupling the endergonic movement of ions with a second, spontaneous process with a large negative ∆G such as the exergonic hydrolysis of ATP (see p. 87). [Note: In the absence of enzymes, ATP is a stable molecule because its hydrolysis has a high Ea.] Figure 6.4 shows a mechanical model of energy coupling. The simplest example of energy coupling in biologic reactions occurs when the energy-requiring and the energy-yielding reactions share a common intermediate. A. Common intermediates Two chemical reactions have a common intermediate when they occur sequentially in that the product of the first reaction is a substrate for the second. For example, given the reactions


Biochemistry_Lippinco

Biochemistry_Lippinco. Two chemical reactions have a common intermediate when they occur sequentially in that the product of the first reaction is a substrate for the second. For example, given the reactions D is the common intermediate and can serve as a carrier of chemical energy between the two reactions. [Note: The intermediate may be linked to an enzyme.] Many coupled reactions use ATP to generate a common intermediate. These reactions may involve the transfer of a phosphate group from ATP to another molecule. Other reactions involve the transfer of phosphate from an energy-rich intermediate to adenosine diphosphate (ADP), forming ATP. B. Energy carried by ATP


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Biochemistry_Lippinco. B. Energy carried by ATP ATP consists of a molecule of adenosine (adenine + ribose) to which three phosphate groups are attached (Fig. 6.5). Removal of one phosphate produces ADP, and removal of two phosphates produces adenosine monophosphate (AMP). For ATP, the ∆G0 of hydrolysis is approximately – 7.3 kcal/mol for each of the two terminal phosphate groups. Because of this large negative ∆G0 of hydrolysis, ATP is called a high-energy phosphate compound. [Note: Adenine nucleotides are interconverted (2 ADP ⇄ ATP + AMP) by adenylate kinase.] V. ELECTRON TRANSPORT CHAIN


Biochemistry_Lippinco

Biochemistry_Lippinco. Energy-rich molecules, such as glucose, are metabolized by a series of oxidation reactions ultimately yielding carbon dioxide and water (H2O), as shown in Figure 6.6. The metabolic intermediates of these reactions donate electrons to specific coenzymes, nicotinamide adenine dinucleotide (NAD+) and flavin adenine dinucleotide (FAD), to form the energy-rich reduced forms, NADH and FADH2. These reduced coenzymes can, in turn, each donate a pair of electrons to a specialized set of electron carriers, collectively called the electron transport chain (ETC), described in this section. As electrons are passed down the ETC, they lose much of their free energy. This energy is used to move H+ across the inner mitochondrial membrane, creating a H+ gradient that drives the production of ATP from ADP and inorganic phosphate (Pi), described on p. 77. The coupling of electron transport with ATP synthesis is called oxidative phosphorylation, sometimes denoted as OXPHOS. It proceeds continuously in all


Biochemistry_Lippinco

Biochemistry_Lippinco. inorganic phosphate (Pi), described on p. 77. The coupling of electron transport with ATP synthesis is called oxidative phosphorylation, sometimes denoted as OXPHOS. It proceeds continuously in all tissues that contain mitochondria. [Note: The free energy not trapped as ATP is used to drive ancillary reactions such as transport of calcium ions into mitochondria and to generate heat.]


Biochemistry_Lippinco

Biochemistry_Lippinco. A. Mitochondrial electron transport chain The ETC (except for cytochrome c, see p. 75) is located in the inner mitochondrial membrane and is the final common pathway by which electrons derived from different fuels of the body flow to oxygen (O2), reducing it to H2O (see Fig. 6.6).


Biochemistry_Lippinco

Biochemistry_Lippinco. 1. Mitochondrial membranes: The mitochondrion contains an outer and an inner membrane separated by the intermembrane space. Although the outer membrane contains special channels (formed by the protein porin), making it freely permeable to most ions and small molecules, the inner membrane is a specialized structure that is impermeable to most small ions, including H+, and small molecules such as ATP, ADP, pyruvate, and other metabolites important to mitochondrial function (Fig. 6.7). Specialized carriers or transport systems are required to move ions or molecules across this membrane. The inner mitochondrial membrane is unusually rich in proteins, over half of which are directly involved in oxidative phosphorylation. It also contains convolutions, called cristae, which greatly increase its surface area.


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Biochemistry_Lippinco. 2. Mitochondrial matrix: The gel-like solution of the matrix (interior) of mitochondria is also rich in proteins. These include the enzymes responsible for the oxidation of pyruvate, amino acids, and fatty acids (by β-oxidation) as well as those of the tricarboxylic acid (TCA) cycle. The synthesis of glucose, urea, and heme occurs partially in the matrix of mitochondria. In addition, the matrix contains NAD+ and FAD (the oxidized forms of the two coenzymes that are required as electron acceptors), and ADP and Pi, which are used to produce ATP. [Note: The matrix also contains mitochondrial deoxyribonucleic acid (mtDNA), ribonucleic acid (mtRNA), and ribosomes.] B. Organization


Biochemistry_Lippinco

Biochemistry_Lippinco. B. Organization The inner mitochondrial membrane contains four separate protein complexes, called Complexes I, II, III, and IV that each contain part of the ETC (Fig. 6.8). These complexes accept or donate electrons to the relatively mobile electron carrier coenzyme Q (CoQ) and cytochrome c. Each carrier in the ETC can receive electrons from an electron donor and can subsequently donate electrons to the next acceptor in the chain. The electrons ultimately combine with O2 and H+ to form H2O. This requirement for O2 makes the electron transport process the respiratory chain, which accounts for the greatest portion of the body’s use of O2. C. Reactions


Biochemistry_Lippinco

Biochemistry_Lippinco. C. Reactions With the exception of CoQ, which is a lipid-soluble quinone, all members of the ETC are proteins. These may function as enzymes as is the case with the flavin-containing dehydrogenases, may contain iron as part of an iron-sulfur (Fe-S) center, may contain iron as part of the porphyrin prosthetic group of heme as in the cytochromes, or may contain copper (Cu) as does the cytochrome a + a3 complex. 1. NADH formation: NAD+ is reduced to NADH by dehydrogenases that remove two hydrogen atoms from their substrate. [Note: For examples of these reactions, see the discussion of the dehydrogenases of the TCA cycle, p. 112.] Both electrons but only one H+ (that is, a hydride ion [:H−]) are transferred to the NAD+, forming NADH plus a free H+. 2.


Biochemistry_Lippinco

Biochemistry_Lippinco. 2. NADH dehydrogenase: The free H+ plus the hydride ion carried by NADH are transferred to NADH dehydrogenase, a protein complex (Complex I) embedded in the inner mitochondrial membrane. Complex I has a tightly bound molecule of flavin mononucleotide (FMN), a coenzyme structurally related to FAD (see Fig. 28.15, p. 384) that accepts the two hydrogen atoms (2 electrons + 2 H+), becoming FMNH2. NADH dehydrogenase also contains peptide subunits with Fe-S centers (Fig. 6.9). At Complex I, electrons move from NADH to FMN to the iron of the Fe-S centers and then to CoQ. As electrons flow, they lose energy. This energy is used to pump four H+ across the inner mitochondrial membrane, from the matrix to the intermembrane space. 3. Succinate dehydrogenase: At Complex II, electrons from the succinate dehydrogenase–catalyzed oxidation of succinate to fumarate move from the coenzyme, FADH2, to an Fe-S protein, and then to CoQ. [Note:


Biochemistry_Lippinco

Biochemistry_Lippinco. Because no energy is lost in this process, no H+ are pumped at Complex II.] 4. Coenzyme Q: CoQ is a quinone derivative with a long, hydrophobic isoprenoid tail. It is made from an intermediate of cholesterol synthesis (see p. 221). [Note: It is also called ubiquinone because it is ubiquitous in biologic systems.] CoQ is a mobile electron carrier and can accept electrons from NADH dehydrogenase (Complex I), from succinate dehydrogenase (Complex II) and from other mitochondrial dehydrogenases, such as glycerol 3-phosphate dehydrogenase (see p. 80) and acyl CoA dehydrogenases (see p. 192). CoQ transfers electrons to Complex III (cytochrome bc1). Thus, a function of CoQ is to link the flavoprotein dehydrogenases to the cytochromes. 5.