Distribution of the thiamin diphosphate C(2)-proton during catalysis of acetaldehyde formation by brewers' yeast pyruvate decarboxylase.
Distribution of the thiamin diphosphate C(2)-proton during catalysis of acetaldehyde formation by brewers' yeast pyruvate decarboxylase.
复制标题
啤酒酵母丙酮酸脱羧酶催化乙醛形成过程中二磷酸硫胺素 C(2)-质子的分布。
DOI：
10.1021/bi00043a005
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发表时间：
1995
期刊：
影响因子：
2.9
通讯作者：
Washabaugh,MW
中科院分区：
文献类型：
--
作者：
Harris,TK;Washabaugh,MW
修订稿于 1995 年 8 月 2 日收到® 摘要：在单周转条件下测定了在卡尔斯伯酵母的丙酮酸脱羧酶同工酶（PDC；EC 4.1. 1.1）催化的丙酮酸形成乙醛的反应过程中，酶结合的[噻唑-2-T]硫胺素二磷酸（TDP）衍生的氚的分布([E]>[S]) 在非底物变构效应物丙酮酰胺存在的情况下。 [1-L]乙醛产物和溶剂([LJHaO)的比放射性分别为PDC结合的[噻唑-2-T] TDP的初始比放射性的43±4%和54±2%，并且与单周转反应的程度无关。提取的 C (2)-氢很少 (< 3%) 或没有返回到 PDC 结合的 TDP 的 C (2) 位置。这提供了证据，表明提取的C(2)-氢参与了PDC结合的中间体2-(1-羟乙基)硫胺素二磷酸(HETDP)的C(a)位的特异性质子化，该中间体被裂解形成[1-L]乙醛和PDC结合的[噻唑-2-H]TDP。 C(2)衍生的氚部分交换到溶剂中需要(1)氢从C(2)转移到催化碱，其中共轭催化酸部分屏蔽与溶剂的氢交换，(2)共轭催化酸将C(2)衍生的氢转移到HETDP的C(a)位置，和(3)氢转移到C(2)以再生辅酶，直接从溶剂或从第二催化酸发生与溶剂进行快速氢交换的酶。在不存在底物的情况下，在 PDC 结合的 TDP 中观察到的 C (2)-氢交换的速率常数 fc0bsd= 1.4 min-1 对应于活性位点 17.5 处的 C (2)-H 的 p 值。丙酮酸脱羧酶 (PDC) 1（2-含氧酸羧基裂解酶；EC 4.1. 1.1）是二磷酸硫胺素 (TDP，1a) 依赖性的催化丙酮酸不可逆非氧化脱羧形成乙醛的酶（方案 1）（Alvarez 等人，1991、1995；Crane 等人，1993）。 PDC 还催化两个乙醛分子之间的羟醛型缩合反应，形成酮醇乙偶姻（Stivers & Washabaugh，1993）。衍生自TDP (Ia)的C(2)-内立德2和衍生自2-(1-羟乙基)硫胺素二磷酸（HETDP，4a)的C(a)-碳负离子/烯胺3已涉及由几种TDP依赖性酶催化的反应（Kluger，1992)。我们感兴趣的是影响动势垒的因素以及 PDC 如何催化质子在 TDP 的 C (2) 位置和 HETDP 的 C (a) 位置之间的转移。
Revised Manuscript Received August 2, 1995® abstract: The distribution of tritium derived from enzyme-bound [thiazole-2-T] thiamin diphosphate (TDP) during the reaction of pyruvate to form acetaldehyde catalyzed by pyruvate decarboxylase isozymes (PDC; EC 4.1. 1.1) from Saccharomyces carlsbergensis was determined under single-turnover conditions ([E]>[S]) in the presence of the nonsubstrate allosteric effector pyruvamide. The specific radioactivity of the [1-L] acetaldehyde product and solvent ([LJHaO) was 43±4% and 54±2%, respectively, of the initial specific radioactivity of PDC-bound [thiazole-2-T] TDP and was independent of the extent of the single-turnover reaction. There is little (< 3%) or no return of the abstracted C (2)-hydron to the C (2) position of PDC-bound TDP. This provides evidence that the abstracted C (2)-hydron is involved in the specific protonation of the C (a) position of the PDC-bound intermediate 2-(1-hydroxyethyl) thiamin diphosphate (HETDP), which is cleaved to form [1-L] acetaldehyde and PDC-bound [thiazole-2-H] TDP. The partial exchange of C (2)-derived tritium into solvent requires that (1) hydron transfer from C (2) occurs to a catalytic base in which the conjugate catalytic acid is partially shielded from hydron exchange with the solvent,(2) the conjugate catalytic acid transfers the C (2)-derived hydron to the C (a) position of HETDP, and (3) hydron transfer to C (2) to regenerate the coenzyme occurs either from solvent directly or from a second catalytic acid of the enzyme that undergoes rapid hydron exchange with the solvent. The observed rate constant fc0bsd= 1.4 min-1 for C (2)-hydron exchange in PDC-bound TDP in the absence of substrate corresponds to a p value for C (2)-H at the active site of 17.5.Pyruvate decarboxylase (PDC) 1 (2-oxo-acid carboxy-lyase; EC 4.1. 1.1) is a thiamin diphosphate (TDP, la) dependent enzyme that catalyzes the irreversible nonoxidative decar-boxylation of pyruvate to form acetaldehyde (Scheme 1)(Alvarez et al., 1991, 1995; Crane et al., 1993). PDC also catalyzes an aldol-type condensation reaction between two molecules of acetaldehyde to form the-ketol acetoin (Stivers & Washabaugh, 1993). The C (2)-ylide 2 derived from TDP (la) and the C (a)-carbanion/enamine 3 derived from 2-(l-hydroxyethyl) thiamin diphosphate (HETDP, 4a) have been implicated in reactions catalyzed by several TDP-dependent enzymes (Kluger, 1992). We are interested in the factor (s) that contribute to the kinetic barrier (s) and how PDC catalyzes proton transfer to and from the C (2) position of TDP and the C (a) position of HETDP.