CATALYTIC POWER OF PYRUVATE DECARBOXYLASE - RATE-LIMITING EVENTS AND MICROSCOPIC RATE CONSTANTS FROM PRIMARY CARBON AND SECONDARY HYDROGEN ISOTOPE EFFECTS
CATALYTIC POWER OF PYRUVATE DECARBOXYLASE - RATE-LIMITING EVENTS AND MICROSCOPIC RATE CONSTANTS FROM PRIMARY CARBON AND SECONDARY HYDROGEN ISOTOPE EFFECTS
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DOI：
10.1021/ja00022a030
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发表时间：
1991-10-23
影响因子：
15
通讯作者：
SCHOWEN, RL
中科院分区：
文献类型：
--
作者：
ALVAREZ, FJ;ERMER, J;SCHOWEN, RL
对于每个稳态动力学参数k/A，测定了卡尔氏酵母（EC www.example.com）的硫胺素二磷酸依赖性丙酮酸脱羧酶4.1.1.1对丙酮酸、丙酮酸-1-C-13、丙酮酸-2-C-13和丙酮酸-3-d3作用的同位素效应（[轻同位素底物的速率常数]/[重同位素底物的速率常数]（丙酮酸的二级）、k/B（丙酮酸的一级）和k（丙酮酸的零级）。 1-C-13效应为1.008 +/- 0.010（k/A）、1.013 +/- 0.024（k/B）和1.024 +/- 0.006（k）。 2-C-13的影响为1.013 +/- 0.009（k/A）、0.951 +/- 0.020（k/B）和1.039 +/- 0.004（k）。 3-d3效应为0.883 +/- 0.013（k/A）、0.881 +/- 0.026（k/B）和1.057 +/- 0.005（k）。 2-氧代丁酸酯和2-氧代丁酸酯-3-d2的效应为0.951 +/- 0.012（k/A）、0.821 +/- 0.096（k/B）和1.057 +/- 0.005（k）。 丙酮酸脱羧酶是已知的滞后激活的底物，丙酮酸结合的调节位点的解离常数为8 mM，并产生单分子激活（0.46 s-1）和失活（0.033 s-1）。 同位素效应导致底物与催化位点结合的速率常数为8.2 × 10（4）M-1 s-1，底物离开催化位点的速率常数为120 s-1，脱羧速率常数为640 s-1，产物释放速率常数为640 s-1。 丙酮酸脱羧酶在pH6.2，30 ℃时可使单独使用硫胺素的丙酮酸脱羧速率增加3 × 10（12）倍。 在这些条件下，活化的酶和丙酮酸在脱羧之前转化为酶物质比特异性碱催化的硫胺素加到丙酮酸快4 × 10（12）倍。 脱羧作用前的酶物质回复为活化酶和游离丙酮酸的速度比硫胺素和丙酮酸的加合物在特异性碱催化下回复为硫胺素和游离丙酮酸的速度快6 × 10（9）倍。 酶促脱羧比硫胺素和丙酮酸加合物的脱羧快10（7）倍。
Isotope effects ([rate constant for light isotopic substrate]/[rate constant for heavy isotopic substrate]) for the action of the thiamin diphosphate dependent pyruvate decarboxylase of Saccharomyces carlsbergensis (EC 4.1.1.1) on pyruvate, pyruvate-1-C-13, pyruvate-2-C-13, and pyruvate-3-d3 have been determined for each of the steady-state kinetic parameters k/A (second-order in pyruvate), k/B (first-order in pyruvate), and k (zero-order in pyruvate). The 1-C-13 effects are 1.008 +/- 0.010 (k/A), 1.013 +/- 0.024 (k/B), and 1.024 +/- 0.006 (k). The 2-C-13 effects are 1.013 +/- 0.009 (k/A), 0.951 +/- 0.020 (k/B), and 1.039 +/- 0.004 (k). The 3-d3 effects are 0.883 +/- 0.013 (k/A), 0.881 +/- 0.026 (k/B), and 1.057 +/- 0.005 (k). Effects with 2-oxobutanoate and 2-oxobutanoate-3-d2 are 0.951 +/- 0.012 (k/A), 0.821 +/- 0.096 (k/B), and 1.057 +/- 0.005 (k). Pyruvate decarboxylase was already known to be hysteretically activated by the substrate, with pyruvate binding to the regulatory site with dissociation constant 8 mM and producing unimolecular activation (0.46 s-1) and deactivation (0.033 s-1). The isotope effects lead to rate constants for substrate binding to the catalytic site of 8.2 x 10(4) M-1 s-1, for substrate departure from the catalytic site of 120 s-1, for decarboxylation of 640 s-1, and for product release of 640 s-1. Pyruvate decarboxylase increases the rate of decarboxylation of pyruvate by thiamin alone by a factor of 3 x 10(12) at pH 6.2, 30-degrees-C. Under these conditions, conversion of activated enzyme and pyruvate to the enzymic species preceding decarboxylation is 4 x 10(12) times faster than the specific-base-catalyzed addition of thiamin to pyruvate. The enzymic species preceding decarboxylation reverts to activated enzyme and free pyruvate 6 x 10(9) times faster than the specific-base-catalyzed reversion of the adduct of thiamin and pyruvate to thiamin and free pyruvate. Enzymic decarboxylation is 10(7) times faster than decarboxylation of the adduct of thiamin and pyruvate.