INACTIVATION OF DIMERIC D-AMINO-ACID TRANSAMINASE BY A NORMAL SUBSTRATE THROUGH FORMATION OF AN UNPRODUCTIVE COENZYME ADDUCT IN ONE SUBUNIT

D-Amino acid transaminase, which contains pyridoxal 5'-phosphate (vitamin B6) as coenzyme, catalyzes the formation Of D-alanine and D-glutamate from their corresponding a-keto acids; these D-amino acids are required for bacterial cell wall biosynthesis. Under conditions usually used for kinetic assay of enzyme activity, i.e., short incubation times with dilute enzyme concentrations, D-alanine behaves as one of the best substrates. However, the enzyme slowly loses activity over a period of hours when exposed to substrates, intermediates, and products at equilibrium. The rate of inactivation is dependent on enzyme concentration but independent of substrate concentration greater than K(m) values. Continuous removal of the product pyruvate by enzymic reduction precludes the establishment of equilibrium and prevents inactivation. The formation of small but detectable amounts of a quinonoid intermediate absorbing at 493 nm is proportional to inactivation. Studies with [C-14]-D-alanine labeled on different carbon atoms indicate that the alpha-carboxyl group of the substrate is absent in the inactive enzyme; such decarboxylation is not a usual function of this enzyme. The inactive transaminase contains 1.1 mol of [C-14]-D-alanine-derived adduct per mole of dimeric enzyme; this finding is consistent with the 50% reduction in the fluorescence intensity at 390 nm (due to the PMP form of the coenzyme) for the inactive enzyme. Thus, inactivation of one subunit of the dimeric enzyme renders the entire molecule inactive. Inactivation may occur when a coenzyme intermediate, perhaps the ketimine, is slowly decarboxylated and then undergoes a conformational change from its catalytically competent location. Evidence indicates that acetaldehyde is the adduct attached to the coenzyme and not to the protein in the inactive enzyme. The activity and spectral properties of the native enzyme are restored upon treatment of the inactive enzyme at slightly acidic pH. These findings may have ramifications in terms of a slow decrease in enzyme function under physiological conditions.