Extent of proton transfer in the transition states of the reaction catalyzed by the delta 5-3-ketosteroid isomerase of Comamonas (Pseudomonas) testosteroni: site-specific replacement of the active site base, aspartate 38, by the weaker base alanine-3-sulf
Extent of proton transfer in the transition states of the reaction catalyzed by the delta 5-3-ketosteroid isomerase of Comamonas (Pseudomonas) testosteroni: site-specific replacement of the active site base, aspartate 38, by the weaker base alanine-3-sulf
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睾丸酮丛毛单胞菌（假单胞菌）的 δ 5-3-酮类固醇异构酶催化的反应过渡态中质子转移的程度：用较弱的碱丙氨酸-3-硫对活性位点碱基天冬氨酸 38 进行位点特异性替换
DOI：
10.1021/bi00175a041
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发表时间：
1994
期刊：
影响因子：
2.9
通讯作者：
Benisek,WF
中科院分区：
文献类型：
--
作者：
Holman,CM;Benisek,WF
1993年12月6日收到的修订版Mandalpt ®摘要：以前对睾丸酮丛毛单胞菌（假单胞菌）类固醇异构酶机制的研究已经确定天冬氨酸38作为质子转运蛋白，将底物的4/3质子转移到产物的6 π位置。因此，天冬氨酸38在底物的去质子化中起碱的作用，以形成二烯醇或二烯醇化物中间体，其然后在C-6/3处从质子化的天冬氨酸38经历再质子化，以得到产物。我们试图通过改变天冬氨酸38的pKf来表征质子转移的过渡态，然后确定改变对酶动力学的影响。pKf的改变是通过用硫取代天冬氨酸38的羧基碳来实现的，这种变化将羧酸酯基团转化为碱性小得多的亚磺酸酯基团。采用布朗斯台德催化理论，适用于异构酶机制的各个步骤，我们发现，在烯醇化反应步骤中的质子转移到天冬氨酸38是在过渡态先进。在次丁酮化步骤中，当达到过渡态时，来自天冬氨酸38的质子转移才刚刚开始。采用定点突变和化学修饰相结合的方法，已经构建了一系列在38位具有替代碱基的突变型KSIs：Asp-38变为Glu（D38 E）、His（D38 H）和S ′-（羧甲基）半胱氨酸（D38 CMC）。D38 H和D38 E突变体都具有显著的异构酶活性，而D38 CMC基本上是惰性的。从动力学实验的结果，它是可能得到一个定性的想法的敏感性酶的催化能力的位置负责质子转移的基地。
Revised Manuscript Received December 6, 1993® abstract: Previous studies of the mechanism of the steroid isomerase of Comamonas (Pseudomonas) testosteroni have identified aspartate 38 as the proton porter which transfers the substrate’s 4/3 proton to the 6ß position of the product. Consequently, aspartate 38 functions as a base in the deprotonation of the substrate to form a dienol or dienolate intermediate, which then undergoes reprotonation from protonated aspartate 38 at C-6/3 to give the product. We have tried to characterize the transition states for the proton transfers by altering the pKf of aspartate 38 and then determining the effect of the alteration on the kinetics of the enzyme. Alteration of the pKf was accomplished by replacement of the carboxyl carbon of aspartate 38 by sulfur, a change which converts the carboxylate group to the much less basic sulfinate group. Employing Bronsted catalysis theory as applied to the individual steps of the isomerase mechanism, we find that in the enolization step of the reaction proton transfer to aspartate 38 is well advanced in the transition state. In thesubsequent ketonization step, proton transfer from aspartate 38 has barely started when that transition state is reached. A series of mutant KSIs with alternative bases at position 38 have been constructed using a combination of site-directed mutagenesisand chemical modification: Asp-38 to Glu (D38E), His (D38H), and S'-(carboxymethyl) cysteine (D38CMC). While the D38H and D38E mutants bothretain significant isomerase activity, D38CMC is essentially inert. From the results of kinetic experiments it is possible to get a qualitative idea of the sensitivity of the enzyme’s catalytic ability to the location of the base responsible for proton transfer.