Maupin C Mark, Zheng Jiayin, Tu Chingkuang, McKenna Robert, Silverman David N, Voth Gregory A
Center for Biophysical Modeling and Simulation and the Department of Chemistry, University of Utah, Salt Lake City, Utah 84112, USA.
Biochemistry. 2009 Aug 25;48(33):7996-8005. doi: 10.1021/bi901037u.
The rate-limiting proton transfer (PT) event in the site-specific mutant N67L of human carbonic anhydrase II (HCA II) has been examined by kinetic, X-ray, and simulation approaches. The X-ray crystallography studies, which were previously reported, and molecular dynamics (MD) simulations indicate that the proton shuttling residue, His64, predominantly resides in the outward orientation with a significant disruption of the ordered water in the active site for the dehydration pathway. While disorder is seen in the active-site water, water cluster analysis indicates that the N67L mutant may form water clusters similar to those seen in the wild-type (WT). For the hydration pathway of the enzyme, the active site water cluster analysis reveals an inability of the N67L mutant to stabilize water clusters when His64 is in the inward orientation, thereby favoring PT when His64 is in the outward orientation. The preference of the N67L mutant to carry out the PT when His64 is in the outward orientation for both the hydration and dehydration pathway is reasoned to be the main cause of the observed reduction in the overall rate. To probe the mechanism of PT, solvent H/D kinetic isotope effects (KIEs) were experimentally studied with catalysis measured by the exchange of (18)O between CO(2) and water. The values obtained from the KIEs were determined as a function of the deuterium content of solvent, using the proton inventory method. No differences were detected in the overarching mechanism of PT between WT and N67L HCA II, despite changes in the active-site water structure and/or the orientation of His64.
已通过动力学、X射线和模拟方法研究了人碳酸酐酶II（HCA II）位点特异性突变体N67L中的限速质子转移（PT）事件。先前报道的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主要以向外的方向存在，这对脱水途径活性位点中的有序水有显著破坏。虽然在活性位点的水中观察到无序，但水簇分析表明N67L突变体可能形成与野生型（WT）中类似的水簇。对于该酶的水合途径，活性位点水簇分析表明，当His64向内时，N67L突变体无法稳定水簇，因此当His64向外时有利于质子转移。N67L突变体在His64向外时对水合和脱水途径都进行质子转移的偏好被认为是观察到的总体速率降低的主要原因。为了探究质子转移的机制，通过测量CO₂与水之间的¹⁸O交换来进行催化，对溶剂H/D动力学同位素效应（KIEs）进行了实验研究。使用质子总量法，将从KIEs获得的值确定为溶剂氘含量的函数。尽管活性位点水结构和/或His64的方向发生了变化，但在WT和N67L HCA II之间未检测到质子转移总体机制的差异。
