 Experimental confirmation of the deprotonation, carbanion formation, and the rate-limiting step of protonation causing cleavage means this is an E1cb mechanism.The most recent data suggest that the catalytic acid is His171, which was previously thought to be the catalytic base, and that somewhat unusually it is a serine at position 295 acts as the catalytic base.The cleavage of adenylosuccinate to AMP and fumarate is an ordered uni-bi mechanism, which means that after cleavage the fumarate leaves the active site before the AMP does.