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首页> 外文期刊>Biochemistry >Dependence of the phosphorylation of alkaline phosphatase by phosphate monoesters on the pKa of the leaving group.
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Dependence of the phosphorylation of alkaline phosphatase by phosphate monoesters on the pKa of the leaving group.

机译：磷酸单酯对碱性磷酸酶的磷酸化依赖于离去基团的pKa。

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摘要

The hydrolysis and transphosphorylation reactions of a series of phosphate monoesters, ROPO3(2)-(R = 2,4-dinitrophenyl, 4-nitrophenyl, phenyl, glucose-1, glycerol-1, methyl, ethyl, and dodecyl), catalyzed by Escherichia coli alkaline phosphatase and a mutant enzyme, Ser102Cys, have been studied at alkaline pH using the rates of change in the 31P NMR signals of substrate, the hydrolysis product (inorganic phosphate), and the transphosphorylation product (O-Tris phosphate) as the assay. The kcat at pH 8.0 for the wild-type enzyme is approximately 30 s-1 and is independent of the nature of the R group, when the pKa of the leaving group is < 10. Under these conditions the rate of phosphorylation is much faster than dissociation of inorganic phosphate, 15-60 s-1. If the pKa of the leaving group is between 10 and 15, phosphorylation and dissociation of the product phosphate both contribute to the rate limit. If the pKa of the leaving group is > 15, phosphorylation is rate limiting. A Bronsted plot of log kcat vs pKa of the leaving group for those substrates for which phosphorylation is rate limiting yields a beta lg of approximately -0.6. In contrast to the wild-type enzyme, the log kcat values for the S102C mutant enzyme catalyzing the hydrolysis of phosphate esters are linearly dependent on the pKa's of the leaving group throughout the range of pKa from 4 to 16. Phosphorylation of C102 is the rate controlling step, and kcat is independent of the Tris concentration as predicted for rate limiting phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)
机译：一系列磷酸单酯ROPO3（2）-（R = 2,4-二硝基苯基，4-硝基苯基，苯基，葡萄糖-1，甘油-1，甲基，乙基和十二烷基）的水解和反磷酸化反应大肠杆菌碱性磷酸酶和突变酶Ser102Cys已在碱性pH下使用底物，水解产物（无机磷酸酯）和反磷酸化产物（O-Tris磷酸酯）的31P NMR信号变化率进行了研究。分析。当离去基团的pKa <10时，野生型酶在pH 8.0时的kcat约为30 s-1，并且与R基团的性质无关。在这些条件下，磷酸化的速率比无机磷酸盐解离，15-60 s-1。如果离去基团的pKa在10至15之间，则产物磷酸酯的磷酸化和解离均会影响速率极限。如果离去基团的pKa＞ 15，则磷酸化是限速的。对于那些磷酸化是限速的底物，log kcat对离去基团的pKa的布朗斯台德图产生的βlg约为-0.6。与野生型酶相反，在pKa从4到16的整个范围内，催化磷酸酯水解的S102C突变酶的log kcat值线性依赖于离去基团的pKa。控制步骤，并且kcat与限速磷酸化所预测的Tris浓度无关。（摘要截短为250字）

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