Investigation of the enzymatic mechanism of yeast orotidine-5'-monophosphate decarboxylase using 13C kinetic isotope effects.
Investigation of the enzymatic mechanism of yeast orotidine-5'-monophosphate decarboxylase using 13C kinetic isotope effects.
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利用 13C 动力学同位素效应研究酵母乳清苷-5-单磷酸脱羧酶的酶促机制。
DOI：
10.1021/bi00239a020
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发表时间：
1991
期刊：
影响因子：
2.9
通讯作者：
Jones,ME
中科院分区：
文献类型：
--
作者：
Smiley,JA;Paneth,P;O'Leary,MH;Bell,JB;Jones,ME
1991年4月9日收到的修订手稿摘要：来自酿酒酵母的乳清酸核苷-S '-单磷酸脱羧酶（ODCase）在25 C、pH 6.8下显示出观察到的13 C动力学同位素效应为1.0247±0.0008。所观察到的同位素效应对反应介质中的变化敏感，例如pH、温度或甘油含量。在pH4.0，25 ℃下测得的值为1.0494±0.0006，温度或甘油对该值没有显著影响，因此在这些条件下显然观察到反应的固有同位素效应，脱羧几乎完全是速率决定性的。这些数据需要具有自由可逆结合的催化机制和其中通过脱羧之前的化学步骤对总速率做出非常有限的贡献的催化机制; Beak和Siegel的两性离子交换机制[Beak，P. & Siegel，B.等（1976）J. Am. Chem.Soc.98，3601-3606]，其仅涉及嘧啶环的质子化，就是这样一种机制。利用1.05的固有同位素效应，计算出在pH6.0，25 ℃时ODCase的分配因子小于1.使用该结果进行的定量动力学分析排除了涉及酶亲核试剂与嘧啶环C-5共价连接的酶机制的可能性。所观察到的同位素效应在pH 8.5以上不会上升到本征值;相反，在25 ℃下观察到的同位素效应对pH作图产生不对称曲线，在高pH下在约1.035处达到平台。这些数据与Vmax/Km的pH曲线相结合，符合一个动力学模型，在该模型中，催化所需的酶质子在高pH下滴定，从而为Beak和Siegel（1976）的催化机制提供了证据。鱼藤苷-5 '-单磷酸脱羧酶（ODCase，1 EC 4.1. 1.23），在从头嘧啶生物合成途径的第六步和最后一步中催化OMP转化为UMP。净反应涉及汽车的替代-
Revised Manuscript Received April 9, 1991 abstract: Orotidine-S'-monophosphate decarboxylase (ODCase) from Saccharomyces cerevisiae displays an observed 13C kinetic isotope effect of 1.0247±0.0008 at 25 C, pH 6.8. The observed isotope effect is sensitive to changes in the reaction medium, such as pH, temperature, or glycerol content. The value of 1.0494±0.0006 measured at pH 4.0, 25 C, is not altered significantly by temperature or glycerol, and thus the intrinsic isotope effect for the reaction is apparently beingobserved under these conditions and decarboxylation is almost entirely rate-determining. These data require a catalytic mechanism with freely reversible binding and one in which a very limited contribution to the overall rate is made by chemical steps preceding decarboxylation; the zwitterion mechanism of Beak and Siegel [Beak, P. & Siegel, B.(1976) J. Am. Chem. Soc. 98, 3601-3606], which involves only protonation of the pyrimidine ring, is such a mechanism. With use of an intrinsic isotope effect of 1.05, a partitioning factor of less than unity is calculated for ODCase at pH 6.0, 25 C. A quantitative kinetic analysis using this result excludes the possibility of an enzymatic mechanism involving covalent attachment of an enzyme nucleophile to C-5 of the pyrimidine ring. Theobserved isotope effect does not rise to the intrinsic value above pH 8.5; instead, the observed isotope effects at 25 C plotted against pH yield an asymmetric curve that at high pH plateaus at about 1.035. These data, in conjunctionwith the pH profile of Vmax/Km, fit a kinetic model in which an enzyme proton necessary for catalysis is titrated at highpH, thus providing evidence for the catalytic mechanism of Beak and Siegel (1976).^) rotidine-5'-monophosphate decarboxylase (ODCase, 1 EC 4.1. 1.23), catalyzes the conversion of OMP to UMP in the sixth and final step of the denovo pyrimidine biosynthetic pathway. The net reaction involves substitution of the car-