A kinetic-isotope-effect study of catalysis by Vibrio cholerae neuraminidase.
A kinetic-isotope-effect study of catalysis by Vibrio cholerae neuraminidase.
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霍乱弧菌神经氨酸酶催化的动力学同位素效应研究。
DOI：
10.1042/bj2940653
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发表时间：
1993
期刊：
影响因子：
--
通讯作者：
Sinnott,ML
中科院分区：
文献类型：
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作者：
Guo,X;Sinnott,ML
在pH 5.0条件下，霍乱弧菌神经氨酸酶对7种芳基N-乙酰α-D-神经氨酸的米氏参数与离开基团的pKa有很好的相关性(β1g(V/K)=-0.73，r=-0.93；β1g(V)=-0.25；r=-0.95)。在最适pH为5.0时(1.059+/-0.010)和在最适pH为8.0(1.053+/-0.010)时，对硝基苯苷的动力学同位素效应βD2(V)相同，表明在最适pH下该底物能充分表达同位素效应。在pH值为5.0时，离开18O基团的效应分别为18(V)=1.040+/-0.016和18(V/K)=1.046+/-0.015，各次生氚效应分别为β-ProRD(V)=1.037+/-0.014，β-ProSD(V)=1.018+/-0.015，β-ProRD(V/K)=1.030+/-0.017，β-ProSD(V/K)=1.030+/-0.017。所有的同位素效应和β1g(V/K)值都符合第一个化学步骤，既是酶周转的第一个不可逆步骤，也是酶周转的速率决定步骤，过渡状态是离开基团的质子很少给予，C-O键大部分断裂，有显著的亲核参与，糖环处于基态2C5椅子的构象。β1g(V)值-0.25与所有动力学-同位素效应数据之间的明显冲突可以通过假设苷元环上的pi系统与酶上的阴离子或亲核基团之间的相互作用来解决。
Michaelis-Menten parameters for hydrolysis of seven aryl N-acetyl alpha-D-neuraminides by Vibrio cholerae neuraminidase at pH 5.0 correlate well with the leaving-group pKa (delta pK 3.0; beta 1g (V/K) = -0.73, r = -0.93; beta 1g (V) = -0.25; r = -0.95). The beta-deuterium kinetic-isotope effect, beta D2(V), for the p-nitrophenyl glycoside is the same at the optimum pH of 5.0 (1.059 +/- 0.010) as at pH 8.0 (1.053 +/- 0.010), suggesting that isotope effects are fully expressed with this substrate at the optimum pH. For this substrate at pH 5.0, leaving group 18O effects are 18(V) = 1.040 +/- 0.016 and 18(V/K) = 1.046 +/- 0.015, and individual secondary deuterium effects are beta proRD(V) = 1.037 +/- 0.014, beta proSD(V) = 1.018 +/- 0.015, beta proRD(V/K) = 1.030 +/- 0.017, beta proSD(V/K) = 1.030 +/- 0.017. All isotope effects, and the beta 1g(V/K) value are in accord with the first chemical step being both the first irreversible and the rate-determining step in enzyme turnover, with a transition state in which there is little proton donation to the leaving group, the C-O bond is largely cleaved, there is significant nucleophilic participation, and the sugar ring is in a conformation derived from the ground-state 2C5 chair. The apparent conflict between the beta 1g (V) value of -0.25 with all the kinetic-isotope-effect data can be resolved by the postulation of an interaction between the pi system of the aglycone ring and an anionic or nucleophilic group on the enzyme.