Rapid Kinetic and Isotopic Studies on Dialkylglycine Decarboxylase

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• 作者：Xianzhi Zhou ; Xueguang Jin ; Rohit Medhekar ; Xiangyang Chen ; Thorsten Dieckmann ; and Michael D. Toney
• 刊名：Biochemistry
• 出版年：2001
• 出版时间：February 6, 2001
• 年：2001
• 卷：40
• 期：5
• 页码：1367 - 1377
• 全文大小：167K
• 年卷期：v.40,no.5(February 6, 2001)
• ISSN：1520-4995

文摘

The two half-reactions of the pyridoxal 5'-phosphate (PLP)-dependent enzyme dialkylglycinedecarboxylase (DGD) were studied individually by multiwavelength stopped-flow spectroscopy. Biphasicbehavior was found for the reactions of DGD-PLP, consistent with two coexisting conformations observedin 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 thetransamination half-reaction between DGD-PMP and pyruvate is 75 s^-1. The maximal rate constant forthe transamination half-reaction of the potassium form of DGD-PLP with L-alanine is 24 s^-1. The spectraldata indicate that external aldimine formation with either AIB or L-alanine and DGD-PLP is a rapidequilibrium process, as is ketimine formation from DGD-PMP and pyruvate. Absorption ascribable tothe quinonoid intermediate is not observed in the AIB decarboxylation half-reaction, but is observed inthe dead-time of the stopped-flow in the L-alanine transamination half-reaction. The [1-¹³C]AIB kineticisotope effect (KIE) on k_cat for the steady-state reaction is 1.043 ± 0.003, while a value of 1.042 ± 0.009was 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 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 productformation give ratios of 105 and 14 with L-alanine and isopropylamine as substrates, respectively. Theseresults support a rate-limiting, concerted Cs/gifchars/alpha.gif" BORDER=0>-decarboxylation/C4'-protonation mechanism for the AIBdecarboxylation reaction, and rapid equilibrium quinonoid formation followed by rate-limiting protonationto the ketimine intermediate for the L-alanine transamination half-reaction. Energy profiles for the twohalf-reactions are constructed.