pH studies on the mechanism of the pyridoxal phosphate-dependent dialkylglycine decarboxylase

The pH dependence of the steady-state kinetic parameters for the dialkylglycine decarboxylase-catalyzed decarboxylation-dependent transamination between 2-aminoisobutyrate (AIB) and pyruvate is presented. The pH dependence of methylation and DTNB modification reactions, and spectroscopic properties, is used to augment the assignment of the kinetic pK(a)'s to specific ionizations. The coincidence of pK(a) values (similar to 7.4) observed in k(cat)/K-AIB, 1/K-AIB, K-is for pyruvate, K-PLP, and in absorbance and fluorescence titrations demonstrates that AIB is not a sticky substrate. It furthermore suggests that the decarboxylation step, or a conformational isomerization preceding it, limits the rate of the overall catalytic cycle. Coexisting, kinetically distinguishable conformers of DGD-PLP, originating from an alkali metal ion binding site, were previously demonstrated at pH 8.2 for DGD-PLP (Zhou, X., Toney, M. D. Biochemistry 37, 5761-5769). The pK(a) value of similar to 8.8 observed in k(cat), k(cat)/K-AIB, K-d for K+, spectrometric titrations, and the reaction of DGD-PLP with DTNB is tentatively assigned to the conformational change interconverting the two enzyme forms previously characterized. Three pK(a)'s are observed in pH titrations of the DGD-PLP coenzyme absorbance. Individual spectra for the four ionization states are deconvoluted by fitting lognormal curves. All four ionization states have both ketoenamine and enolimine tautomers present. This and a review of spectral data in the literature lead to the conclusion that the pK(a) of similar to 7.4, which gives the largest spectral changes and controls k(cat)/K-AIB, is not deprotonation of the aldimine nitrogen. Rather, it must be an active site residue whose ionization alters the ratio between ketoenamine and enolimine tautomers.