Structural and mechanistic studies of pyridoxal 5'-phosphate dependent enzymes.
The pyridoxal 5'-phosphate (PLP) analogue 4'-N-(2''-phosphoethyl) pyridoxamine 5'-phosphate (EAP-PMP) has been synthetized. The appenzymes of both E.coli L-glutamic acid decarboxylase (GAD) and cytosolic aspartate aminotransferase have been shown to cleave the pyridoxal analogue with the production of PLP, resulting in reactivation of the enzyme. Two other PLP apoenzymes have been purified and have been demonstrated to be inactive towards EAP-PMP. In order to determine the stereochemical course of the proton abstraction step of the reactivation reaction with E.coli GAD (2R)- and (2S)-[1''-2H1]EAP-PMP and [1''-2H2]EAP-PMP were prepared. A key step in the synthesis of these compounds was a stereospecific glycine CPROB*LEM-proton exchange reaction with the solvent as catalysed by glutamic-pyruvic transaminase. Using these labelled coenzyme analgoues it was then shown that the 1''-pro (R) hydrogen is removed during the reactivation reaction. The stereochemical course of protonation at CPROB*LEM in the quinonoid intermediate derived from the decarboxylation of L-aspartic acid by E.coli GAD was also determined. The reaction was shown to proceed with complete retention of configuration. The collective results of these studies are discussed in terms of the disposition of distal binding groups of substrates at the active site of the enzyme. The kinetics of both the decarboxylation and abortive transamination reactions of E.coli GAD, using L-glutamic acid as the substrate have been investigated and the results have been rationalised.