Catalytic acid-base groups in yeast pyruvate decarboxylase. 2. Insights into the specific roles of D28 and E477 from the rates and stereospecificity of formation of carboligase side products
Catalytic acid-base groups in yeast pyruvate decarboxylase. 2. Insights into the specific roles of D28 and E477 from the rates and stereospecificity of formation of carboligase side products
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DOI：
10.1021/bi002856m
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发表时间：
2001-06-26
期刊：
影响因子：
2.9
通讯作者：
Jordan, F
中科院分区：
文献类型：
--
作者：
Sergienko, EA;Jordan, F
酵母丙酮酸脱羧酶(YPDC)除了形成其代谢产物乙醛外，还可以进行碳寡酶反应，其中中心烯胺中间体与乙醛或丙酮酸反应(而不是通常的质子电泳法)，分别生成乙酸乙酯和乙酰乳酸(通常占总反应的1%)。由于乙醛的生成和碳寡酶通过脱羧基反应的共同机理，对活性中心E4774、D28A和D28N的YPDC变异体生成的碳寡酶产物的速率和立体化学进行了详细的分析。虽然在任一位点上的取代导致乙醛生成的催化效率降低了大约2-3个数量级，但E477Q和D28N变异体的乙醛生成率高于野生型酶。对乙醛和乙醛生成的稳态数据的比较表明，D28A、H114F、H115F和E4774变异株形成乙醛的限速步骤是脱羧基后的步骤。与野生型YPDC和E4774变异体相比，D28A和D28N变异体可以合成主要产物乙酰乳酸。与野生型酶相比，D28A变异体副产物的总生成率较低，这证明D28参与了导致和包括脱羧基的步骤。结果还提供了对所考察的侧链的电离状态的洞察。(R)-乙酸乙酯是由具有比野生型YPDC更大的对映体过剩的变体产生的。(S)-乙酰乳酸是D28取代变异体产生的主要对映体，与相关植物乙酰乳酸合成酶产生的构型相同。
Yeast pyruvate decarboxylase (YPDC), in addition to forming its metabolic product acetaldehyde, can also carry out carboligase reactions in which the central enamine intermediate reacts with acetaldehyde or pyruvate (instead of the usual proton electrophile), resulting in the formation of acetoin and acetolactate, respectively (typically, 1% of the total reaction). Due to the common mechanism shared by the acetaldehyde-forming and carboligase reactions through decarboxylation, a detailed analysis of the rates and stereochemistry of the carboligase products formed by the E4774, D28A, and D28N active center YPDC variants was undertaken. While substitution at either position led to an approximately 2-3 orders of magnitude lower catalytic efficiency in acetaldehyde formation, the rate of acetoin formation by the E477Q and D28N variants was higher than that by wild-type enzyme. Comparison of the steady-state data for acetaldehyde and acetoin formation revealed that the rate-limiting step for acetaldehyde formation by the D28A, H114F, H115F, and E4774 variants is a step post-decarboxylation. In contrast to the wild-type YPDC and the E4774 variant, the D28A and D28N variants could synthesize acetolactate as a major product. The lower overall rate of side-product formation by the D28A variant than wild-type enzyme attests to participation of D28 in steps leading up to and including decarboxylation. The results also provide insight into the state of ionization of the side chains examined. (R)-Acetoin is produced by the variants with greater enantiomeric excess than by wild-type YPDC. (S)-Acetolactate is the predominant enantiomer produced by the D28 substituted variants, the same configuration as produced by the related plant acetolactate synthase.