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

Abstract

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 N^4′-(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 C^α 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.