The Location and Role of Active-Site Bases in PLP-Dependent Decarboxylase Enzymes as Deduced from Stereochemical and Kinetic Studies

Summary

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. Treatment of the inactive apoenzyme with N ^4′-(2″-phosphoethyl)-pyridoxamine 5′-phosphate results in the formation of active holoenzyme via a mechanism in which the 1″-pro-R hydrogen and phosphate are eliminated from the phosphoethyl moiety. 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. It is also demonstrated that the decarboxylase is inactivated by (2R)-serine O-sulphate, as well as by the (2S)enantiomer of the suicide inhibitor, and that inactivation by the (2S)-enantiomer involves C^α-H bond cleavage while inactivation by the (2R)-isomer involves C^α-decarboxylation. Both processes occur on the 4′-re-face of the coenzyme, the opposite face to that utilised in the natural decarboxylation reaction.