The stereochemical course of decarboxylation, transamination and elimination reactions catalysed by Escherichia coli glutamic acid decarboxylase

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Pyridoxal 5′-phosphate dependent Escherichia coli glutamic acid decarboxylase reprotonates the quinonoid intermediate derived from the coenzyme and its natural substrate, (2S)-glutamic acid on the 4′-Si-face of the coenzyme during an abortive decarboxylation–transamination reaction. The enzyme introduces the 3-pro-R hydrogen of β-alanine with retention of configuration during the decarboxylation of (2S)-aspartic acid. In the absence of pyridoxal 5′-phosphate, treatment of the inactive apoenzyme with the inhibitor N4′-(2″-phosphoethyl)pyridoxamine 5′-phosphate results in reactivation through the formation of the active pyridoxal 5′-phosphate holoenzyme complex. During this reaction hydrogen phosphate is eliminated from the phosphoethyl moiety. Using synthetic chirally deuteriated isotopomers of the inhibitor it is demonstrated that the 1-pro-R hydrogen of inhibitor is removed during the reactivation reaction. The results suggest that protonations and deprotonations at Cα of quinonoid intermediates derived from the coenzyme and the substrate occur from the 4′-Si-face of the coenzyme and that the distal binding groups of the substrates and inhibitors occupy similar positions at the active site on the 3′-phenolic group side of the coenzyme.