Treponema denticola cystalysin exhibits significant alanine racemase activity accompanied by transamination: mechanistic implications

To obtain information on the reaction specificity of cystalysin from the spirochaete bacterium Treponema denticola, the interaction with L- and D-alanine has been investigated. Binding of both alanine enantiomers leads to the appearance of an external aldimine absorbing at 429 nm and of a band absorbing at 498 nm, indicative of a quinonoid species. Racemization and transamination reactions were observed to occur with both alanine isomers as substrates. The steady-state kinetic parameters for racemization, k(eat) and K-m, for L-alanine are 1.05 +/- 0.03 s(-1) and 10 +/- 1 mM respectively, whereas those for D-alanine are 1.4 +/- 0.1 s(-1) and 10 +/- 1 mM. During the reaction of cystalysin with L- or D-alanine, a time-dependent loss of beta-elimination activity occurs concomitantly with the conversion of the pyridoxal 5'-phosphate (PLP) coenzyme into pyridoxamine 5'-phosphate (PMP). The catalytic efficiency of the half-transamination Of L-alanine is found to be 5.3 x 10(-5) mM(-1) (.) s(-1), 5-fold higher when compared with that Of D-alanine. The partition ratio between racemization and half-transamination reactions is 2.3 x 10(3) for L-alanine and 1.4 x 10(4) for D-alanine. The pH dependence of the kinetic parameters for both the reactions shows that the enzyme possesses a single ionizing residue with pK values of 6.5-6.6, which must be unprotonated for catalysis. Addition of pyruvate converts the PMP form of the enzyme back into the PLP form and causes the concomitant recovery of beta-elimination activity. In contrast with other PLP enzymes studied so far, but similar to alanine racemases, the apoform of the enzyme abstracted tritium from C4' of both (4'S)- and (4'R)[4'-H-3]PMP in the presence of pyruvate. Together with molecular modelling of the putative binding sites Of L- and D-alanine at the active site of the enzyme, the implications of these studies for the mechanisms of the side reactions catalysed by cystalysin are discussed.