Pre-steady-state kinetic analysis of the reactions of alternate substrates with dialkylglycine decarboxylase.
Pre-steady-state kinetic analysis of the reactions of alternate substrates with dialkylglycine decarboxylase.
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替代底物与二烷基甘氨酸脱羧酶反应的前稳态动力学分析。
DOI：
10.1021/bi972056k
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Toney,MD
中科院分区：
文献类型：
--
作者：
Sun,S;Bagdassarian,CK;Toney,MD
用多波长动力学和整体分析方法研究了几种底物与二烷基甘氨酸脱羧酶的半反应稳态前动力学。研究的底物分为两组： 那些表现出简单的单相动力学的底物和那些表现出双相动力学的底物。根据从全局分析获得的简单动力学和光谱，AIB的半反应速度可能受到脱羧基步骤的限制。在L-丙氨酸和L-氨基丁酸酯的转氨化半反应中，第一种物种的光谱表现出碳离子型喹酮中间体的长波吸收特征。这表明外源乙二胺中间体的形成和C-α质子的提取是快速的。较慢的底物1-苯甘氨酸和1-氨基环己烷-1-羧酸酯的反应可能有外部乙二胺的生成作为速率决定步骤。根据吸收光谱和圆二色谱的变化，2-甲基-2-氨基丙酸甲酯、1-氨基环戊烷-1-羧酸酯、异丙胺和甘氨酸的两相反应都以外加乙二胺为快速可观察步骤。2-甲基-2-氨基丙二酸甲酯与二烷基甘氨酸脱羧酶反应∼的速度是∼的104倍，而在溶液中辅酶的反应速度是AIB的105倍。有人认为，这种自由基反应活性的逆转是由于缓慢的蛋白质构象变化，这是MAM脱羧基的先决条件，随后迅速发生。活性部位结合辅酶的圆二色谱测量提供了支持这一建议的证据。非共价抑制剂丙酮酸或乳酸或共价结合抑制剂1-氨基环丙烷-1-羧酸的结合都会引起辅酶圆二色谱的缓慢变化，定量上与2-甲基-2-氨基丙酸的缓慢脱羧基平行。1-氨基环丙烷-1-羧酸酯和2-甲基-2-氨基丙酸甲酯的混合时间都出现了快速的圆二色谱变化，这表明在这个较长的时间尺度上，外部乙二胺的形成速度很快。
The pre-steady-state kinetics of the half-reactions of several substrates with dialkylglycine decarboxylase are examined by multiwavelength kinetics and global analysis. The substrates examined fall into two groups: those that exhibit simple, monophasic kinetics and those that exhibit biphasic kinetics. The rate of the AIB half-reaction is likely limited by the decarboxylation step based on the simple kinetics and spectra obtained from global analysis. The spectra for the first species in the transamination half-reactions ofl-alanine andl-aminobutyrate show long-wavelength absorption characteristic of a carbanionic quinonoid intermediate. This demonstrates that formation of the external aldimine intermediates and abstraction of the Cαprotons from them are rapid. The reactions of the slower substratesl-phenylglycine and 1-aminocyclohexane-1-carboxylate may have external aldimine formation as the rate-determining step. The biphasic reactions of 2-methyl-2-aminomalonate, 1-aminocyclopentane-1-carboxylate, isopropylamine, and glycine all have external aldimine formation as the rapid observable step, based on the spectral changes observed in absorption and circular dichroism measurements. 2-Methyl-2-aminomalonate reacts ∼104-fold slower than does AIB with dialkylglycine decarboxylase, compared to ∼105-fold faster with coenzyme in solution. It is proposed that this radical reactivity reversal is due to a slow protein conformational change that is a prerequisite to decarboxylation of MAM, which occurs rapidly thereafter. Circular dichroism measurements on active site bound coenzyme provide evidence supporting this proposal. The binding of the noncovalent inhibitors pyruvate or lactate or the covalently binding inhibitor 1-aminocyclopropane-1-carboxylate all induce a slow change in coenzyme circular dichroism that quantitatively parallels the slow decarboxylation of 2-methyl-2-aminomalonate. Fast circular dichroism changes are seen in the mixing time of these measurements for both 1-aminocyclopropane-1-carboxylate and 2-methyl-2-aminomalonate, indicating rapid external aldimine formation on this longer time scale.