pH studies on the mechanism of the pyridoxal phosphate-dependent dialkylglycine decarboxylase.
pH studies on the mechanism of the pyridoxal phosphate-dependent dialkylglycine decarboxylase.
复制标题
磷酸吡哆醛依赖性二烷基甘氨酸脱羧酶机制的pH研究。
DOI：
10.1021/bi981455s
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Toney,MD
中科院分区：
文献类型：
--
作者：
Zhou,X;Toney,MD
二烷基甘氨酸脱羧酶催化的脱羧依赖的2-氨基异丁酸（AIB）和丙酮酸之间的转氨作用的稳态动力学参数的pH依赖性。甲基化和DTNB修饰反应的pH依赖性和光谱性质，用于增加分配的动力学pKa的特定电离。在inkcat/KAIB、1/KAIB、Kisfor丙酮酸、KPLP以及吸光度和荧光滴定中观察到的pKa值（约7.4）的一致性表明AIB不是粘性底物。它还表明，脱羧步骤，或构象异构化之前，限制了整个催化循环的速度。DGD-PLP的共存的、动力学上可区分的构象异构体（源自碱金属离子结合位点）先前在pH 8.2下对于DGD-PLP被证明（Zhou，X.，托尼，M。D.Biochemistry37，5761 - 5769）。在inkcat、kcat/KAIB、K+的Kd、光谱滴定以及DGD-PLP与DTNB的反应中观察到的pKa值为1.88，初步归属于先前表征的两种酶形式相互转化的构象变化。在DGD-PLP辅酶吸光度的pH滴定中观察到三个pKa。通过拟合对数正态曲线对四种电离态的单个光谱进行去卷积。所有四种电离状态都存在酮烯胺和烯醇亚胺互变异构体。这一点和文献中的光谱数据的审查导致的结论是，pKaof 7.4，这给出了最大的光谱变化和控制skcat/KAIB，是不是醛亚胺氮的去质子化。相反，它必须是一个活性位点残基，其电离改变酮烯胺和烯醇亚胺互变异构体之间的比例。
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 pKavalues (∼7.4) observed inkcat/KAIB, 1/KAIB,Kisfor 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.Biochemistry37, 5761−5769). The pKavalue of ∼8.8 observed inkcat,kcat/KAIB,Kdfor 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 pKaof ∼7.4, which gives the largest spectral changes and controlskcat/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.