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首页> 外文期刊>Biochemistry >Effect of Active-site Mutation at Asn67 on the Proton Transfer Mechanism of Human Carbonic Anhydrase II
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Effect of Active-site Mutation at Asn67 on the Proton Transfer Mechanism of Human Carbonic Anhydrase II

机译：Asn67活性位点突变对人类碳酸酐酶II质子转移机制的影响

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摘要

The rate-limiting proton transfer (PT) event in the site-specific mutant N67L of human carbonicnanhydrase II (HCA II) has been examined by kinetic, X-ray, and simulation approaches. The X-rayncrystallography studies, which were previously reported, and molecular dynamics (MD) simulations indicatenthat the proton shuttling residue, His64, predominantly resides in the outward orientation with a significantndisruption of the ordered water in the active site for the dehydration pathway. While disorder is seen in thenactive-site water, water cluster analysis indicates that the N67L mutant may form water clusters similar tonthose seen in the wild-type (WT). For the hydration pathway of the enzyme, the active site water clusternanalysis reveals an inability of the N67L mutant to stabilize water clusters when His64 is in the inwardnorientation, thereby favoring PT when His64 is in the outward orientation. The preference of the N67Lnmutant to carry out the PT when His64 is in the outward orientation for both the hydration and dehydrationnpathway is reasoned to be the main cause of the observed reduction in the overall rate. To probe thenmechanism of PT, solvent H/D kinetic isotope effects (KIEs) were experimentally studied with catalysisnmeasured by the exchange of 18O between CO2 and water. The values obtained from the KIEs werendetermined as a function of the deuterium content of solvent, using the proton inventory method. Nondifferences were detected in the overarching mechanism of PT between WT and N67L HCA II, despitenchanges in the active-site water structure and/or the orientation of His64.
机译：已通过动力学，X射线和模拟方法研究了人类碳酸酐酶II（HCA II）的位点特异性突变体N67L中的限速质子转移（PT）事件。先前已报道的X射线晶体学研究和分子动力学（MD）模拟表明，质子穿梭残基His64主要位于向外方向，在脱水途径的活性位点中有序水有显着破坏。虽然在活动现场的水中发现了紊乱，但水簇分析表明N67L突变体可能形成与野生型（WT）中类似豆蔻糖相似的水簇。对于酶的水合途径，活性位点水簇分析表明，当His64处于向内取向时，N67L突变体无法稳定水簇，从而当His64处于向外取向时，有利于PT。 N67L突变体在His64处于外向时因水合和脱水途径的偏好而进行PT被认为是观察到总速率降低的主要原因。为了探讨PT的机理，通过在CO2与水之间交换18O进行催化的实验研究了溶剂H / D动力学同位素效应（KIEs）。使用质子清单法确定从KIE获得的值与溶剂中氘含量的关系。尽管活动位点水结构和/或His64的方向发生了变化，但WT和N67L HCA II之间的PT总体机制没有差异。

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    《Biochemistry》 |2009年第33期|p.7996-8005|共10页
  • 作者

    C. Mark Maupin Jiayin Zheng Chingkuang Tu Robert McKenna David N. Silverman and Gregory A. Voth;

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    ‡Center for Biophysical Modeling and Simulation and the Department of Chemistry, University of Utah, Salt Lake City, Utah 84112,§Department of Pharmacology and Therapeutics, University of Florida, Gainesville, Florida 32610, and)Department of Biochemistryand Molecular Biology, University of Florida, Gainesville, Florida 32610. ^These authors contributed equally to this work.;

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