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B6db references: 21093112

type Journal Article
authors Zhou, X.; Jin, X.; Medhekar, R.; Chen, X.; Dieckmann, T.; Toney, M. D.
title Rapid kinetic and isotopic studies on dialkylglycine decarboxylase
journal Biochemistry
ui 21093112
year (2001)
volume 40
number 5
pages 1367-77.
keywords Alanine/chemistry
abstract The two half-reactions of the pyridoxal 5'-phosphate (PLP)-dependent enzyme dialkylglycine decarboxylase (DGD) were studied individually by multiwavelength stopped-flow spectroscopy. Biphasic behavior was found for the reactions of DGD-PLP, consistent with two coexisting conformations observed in steady-state kinetics [Zhou, X., and Toney, M. D. (1998) Biochemistry 37, 5761--5769]. The half-reaction kinetic parameters depend on alkali metal ion size in a manner similar to that observed for steady-state kinetic parameters. The fast phase maximal rate constant for the 2-aminoisobutyrate (AIB) decarboxylation half- reaction with the potassium form of DGD-PLP is 25 s(-1), while that for the transamination half-reaction between DGD-PMP and pyruvate is 75 s(- 1). The maximal rate constant for the transamination half-reaction of the potassium form of DGD-PLP with L-alanine is 24 s(-1). The spectral data indicate that external aldimine formation with either AIB or L- alanine and DGD-PLP is a rapid equilibrium process, as is ketimine formation from DGD-PMP and pyruvate. Absorption ascribable to the quinonoid intermediate is not observed in the AIB decarboxylation half- reaction, but is observed in the dead-time of the stopped-flow in the L- alanine transamination half-reaction. The [1-(13)C]AIB kinetic isotope effect (KIE) on k(cat) for the steady-state reaction is 1.043 +/- 0.003, while a value of 1.042 +/- 0.009 was measured for the AIB half- reaction. The secondary KIE measured for the AIB decarboxylation half- reaction with [C4'-(2)H]PLP is 0.92 +/- 0.02. The primary [2-(2)H]-L- alanine KIE on the transamination half-reaction is unity. Small but significant solvent KIEs are observed on k(cat) and k(cat)/K(M) for both substrates, and the proton inventories are linear in each case. NMR measurements of C2--H washout vs product formation give ratios of 105 and 14 with L-alanine and isopropylamine as substrates, respectively. These results support a rate-limiting, concerted C alpha- decarboxylation/C4'-protonation mechanism for the AIB decarboxylation reaction, and rapid equilibrium quinonoid formation followed by rate- limiting protonation to the ketimine intermediate for the L-alanine transamination half-reaction. Energy profiles for the two half- reactions are constructed.
last changed 2002/11/12 16:17

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