Kinetics of the reaction of cyclopropanone hydrate with yeast aldehyde dehydrogenase: a model for enzyme--substrate interaction.
Kinetics of the reaction of cyclopropanone hydrate with yeast aldehyde dehydrogenase: a model for enzyme--substrate interaction.
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环丙酮水合物与酵母醛脱氢酶反应的动力学：酶-底物相互作用的模型。
DOI：
10.1021/bi00559a013
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发表时间：
1980
期刊：
影响因子：
2.9
通讯作者：
Abeles,RH
中科院分区：
文献类型：
--
作者：
Wiseman,JS;Tayrien,G;Abeles,RH
Jeffrey S. Wiseman，* Guy Tayrien，and Robert H. Abeles* 摘要：环丙酮水合物与酵母醛脱氢酶的-SH基团形成硫代半缩酮，类似于由醛底物形成的硫代半缩醛[Wiseman，JS，& Abeles，RH（1979）Biochemistry 18，427-435]，但是环丙酮水合物不能经历氧化，因此充当催化过程的抑制剂。我们研究了水合环丙酮与酵母醛脱氢酶反应的动力学，因为我们相信该反应可作为酶与正常底物相互作用的模型。反应是有序的;烟酰胺腺嘌呤二核苷酸（NAD）首先结合。在结合的热力学和动力学方面都观察到了惊人的结果。三元复合物中NAD和环丙酮水合物结合的平衡常数约为10 - 9 M。环丙酮水合物如此高的结合能的来源尚不清楚。在这两种情况下，在三元复合物的结合是近105比在二元复合物紧。三元复合物也是动力学稳定的。NAD从三元复合物中解离的时间为rl/2~ 6天，即比二元复合物慢108倍。有人认为，构象变化是这种解离的速率限制。环丙酮水合物对NAD的结合和解离速率的影响是该分子的独特性质。用碘乙酰胺将与水合环丙酮反应的-SH基烷基化，不影响NAD与酶的反应。烟酰胺单甘肽（NMN）也是醛脱氢酶的辅因子，它不与环丙酮水合物失活酶形成动力学稳定的复合物，也不影响环丙酮水合物与酶的结合。结果表明，NAD与水合环丙酮的结合是协同的。其中一种的结合导致构象变化，从而增强另一种的结合。NAD中的腺苷S '-一磷酸（AMP）部分在引起这种构象变化中是重要的。催化反应的结果证实了与环丙酮水合物的反应是与正常底物反应的适用模型。在苯甲醛存在下，反应是有序的; NAD首先结合。NAD使苯甲醛的结合增强至少500倍，并且从三元复合物中缓慢解离，如通过脉冲追踪实验所证明的[Rose，I.一、O'Connell，E. L.，Litwin，S.，& Bar-Tana，J.（1974）J.Biol.Chem.249，5163-5168]，而NMN对于三元复合物快速解离（脉冲追踪实验）。在NMN的存在下，苯甲醛的Km比NAD的存在下高200倍。
Jeffrey S. Wiseman,* Guy Tayrien, and Robert H. Abeles* abstract: Cyclopropanone hydrate forms a thiohemiketal with an-SH group of yeast aldehyde dehydrogenase, analogous to the thiohemiacetal formed by aldehyde substrates [Wiseman, JS, & Abeles, RH (1979) Biochemistry 18, 427-435], butcyclopropanone hydrate cannot undergo oxi-dation and, therefore, acts as an inhibitor of the catalytic process. We have investigated the kinetics of the reaction of cyclopropanone hydrate with yeast aldehyde dehydrogenase since we believe this reaction serves as a model for the in-teraction of the enzyme with the normal substrate. The re-action is ordered; nicotinamide adenine dinucleotide (NAD) binds first. Striking results are observed in both the thermodynamics and kinetics of binding. Equilibrium constants for binding of both NAD and cyclopropanone hydrate in the ternary complex are on the orderof 10™ 9 M. The sources of such high binding energy for cyclopropanone hydrate are not clear. In both cases, the binding in the ternary complex is nearly 105 tighter than inthe binary complexes. The ternary complex is also kinetically stable. NAD dissociates from the ternary complex with rl/2~ 6 days, ie, 108-fold slower than from the binary complex. It is argued that a conformational change is rate limiting forthis dissociation. The effect of cyclopropanone hydrate on the binding and dissociation rate of NAD is a unique property of that molecule. Alkylation of the-SH group which reacts with cyclopropanone hydrate by iodoacetamide does not effect the reaction of NAD with en-zyme. Nicotinamide mononucleotide (NMN), which is also a cofactor for aldehyde dehydrogenase, does not form a kinetically stable complex with cyclopropanone hydrate inac-tivated enzyme, nor does it affect the binding of cyclo-propanone hydrate to the enzyme. It is concluded that binding of NAD and cyclopropanone hydrate is cooperative. Binding of one results in a conformational change that enhances binding of the other. The adenosine S'-monophosphate (AMP) portion ofNAD is important in bringing about this conformational change. Results obtainedfor the catalytic reaction confirm that the reaction with cyclopropanone hydrate is an applicable model for the reaction with the normal substrate. In the presence of benzaldehyde, the reaction is ordered; NAD binds first. NAD enhances the binding of benzaldehyde at least 500-fold and dissociates slowly from the ternary complex as demonstrated by pulse-chase experiments [Rose, I. A., O’Connell, E. L., Litwin, S., & Bar-Tana, J.(1974) J. Biol. Chem. 249, 5163-5168] while NMN dissociates rapidly for the ternary complex (pulse-chase experiments). In the presence of NMN, Km for benzaldehyde is 200-fold higher than in the presence of NAD.