Quantitative analysis of the effect of salt concentration on enzymatic catalysis
Quantitative analysis of the effect of salt concentration on enzymatic catalysis
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DOI：
10.1021/ja0164834
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发表时间：
2001-11-21
影响因子：
15
通讯作者：
Raines, RT
中科院分区：
文献类型：
--
作者：
Park, C;Raines, RT
和pH一样，盐浓度对酶催化也有很大的影响。其中，K(CAT)/K-M(MAX)是K(CAT)/K-M=(K(CAT)/K-M)(MAX)/[1+([Na+]/K-M)(MAX)/2的盐浓度，-n‘是log(k(Cat)/K-M)-log[Na+]曲线中线性区域的斜率。N‘的值具有特殊的用途，因为它反映了库仑相互作用对束缚态均匀束缚的贡献。用这个方程分析了盐对核糖核酸酶A催化作用的影响。核糖核酸酶A是一种阳离子酶，催化切割阴离子底物核糖核酸酶，其k(CAT)/K-M值可超过10(9)M-1 S(-1)。Lys7、Arg10和Lys66包括远离活性部位的酶亚基。取代Lys7。Arg10和Lys66与丙氨酸结合后，酶上的电荷减少，n‘值减小。同样，减少底物中的磷酸基团数量会降低n的值。用精氨酸取代关键活性中心残基Lys41产生的催化剂，受到底物到产物的化学转化的限制。这种变化增加了n‘的值，对于对化学过渡态的结合变化更敏感的催化剂来说，这是意料之中的。因此，盐分速率分布的定量分析可以为库仑相互作用在酶催化中的作用提供有价值的见解。
Like pH, salt concentration can have a dramatic effect on enzymatic catalysis. Here, a general equation is derived for the quantitative analysis of salt-rate profiles: k(cat)/K-M = (k(cat)/K-M)(MAX)/[1 + ([Na+]/K-Na(+))(n')], where (k(cat)/K-M)(MAX) is the physical limit of k(cat)/K-M, K-Na(+) is the salt concentration at which k(cat)/K-M = (k(cat)/K-M)(MAX)/2, and -n' is the slope of the linear region in a plot of log(k(cat)/K-M) versus log [Na+]. The value of n' is of special utility, as it reflects the contribution of Coulombic interactions to the uniform binding of the bound states. This equation was used to analyze salt effects on catalysis by ribonuclease A (RNase A), which is a cationic enzyme that catalyzes the cleavage of an anionic substrate, RNA, with k(cat)/K-M values that can exceed 10(9) M-1 s(-1). Lys7, Arg10, and Lys66 comprise enzymic subsites that are remote from the active site. Replacing Lys7. Arg10, and Lys66 with alanine decreases the charge on the enzyme as well as the value of n'. Likewise, decreasing the number of phosphoryl groups in the substrate decreases the value of n. Replacing Lys41, a key active-site residue, with arginine creates a catalyst that is limited by the chemical conversion of substrate to product. This change increases the value of n', as expected for a catalyst that is more sensitive to changes in the binding of the chemical transition state. Hence, the quantitative analysis of salt-rate profiles can provide valuable insight into the role of Coulombic interactions in enzymatic catalysis.