Activities | Families | Sequences | Fold types | References | Help
B6db references: 99107613

type Journal Article
authors Zhou, X.; Toney, M. D.
title pH studies on the mechanism of the pyridoxal phosphate-dependent dialkylglycine decarboxylase
journal Biochemistry
ui 99107613
year (1999)
volume 38
number 1
pages 311-20.
keywords Binding Sites
abstract 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 pKa's to specific ionizations. The coincidence of pKa values (approximately 7.4) observed in kcat/KAIB, 1/KAIB, Kis for pyruvate, KPLP, 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 pKa value of approximately 8.8 observed in kcat, kcat/KAIB, Kd 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 pKa's are observed in pH titrations of the DGD-PLP coenzyme absorbance. Individual spectra for the four ionization states are deconvoluted by fitting log-normal 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 pKa of approximately 7.4, which gives the largest spectral changes and controls kcat/KAIB, 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.
last changed 2002/11/12 16:17

B6db references