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 [1-13C]AIB kinetic isotope effect (KIE) on kcat 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′-2H]PLP is 0.92±0.02. The primary [2-2H]-L-alanine KIE on the transamination half-reaction is unity. Small but significant solvent KIEs are observed on kcat and kcat/KM 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 Cα-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.
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