化学
动力学同位素效应
动能
同位素
小学(天文学)
放射化学
溶剂
化学反应动力学
物理化学
化学动力学
氘
动力学
核物理学
有机化学
物理
量子力学
天文
作者
Ananda K. Ghosh,D. Groom,Vern L. Schramm
摘要
Human sterile alpha motif and HD-domain-containing protein 1 (SAMHD1) is an allosterically regulated dNTP triphosphohydrolase (dNTP + H2O → dNuc + PPPi) involved in deoxynucleotide regulation and DNA repair. We characterized the chemical features of the SAMHD1 transition state for 2′-deoxyadenosine 5′-triphosphate (dATP) hydrolysis by analysis of 18O and 33P primary kinetic isotope effects (KIEs) at the α-phosphoryl of the leaving triphosphate group. The intrinsic KIE values for [5′-18O]dATP of 1.028 ± 0.003 and for [α-33P]dATP of 1.015 ± 0.004 provide insights into the mechanistic details of the SAMHD1 transition state. Solvent 2H2O isotope effects for the hydrolysis of dATP indicate that a single proton is being transferred at the transition state to give a solvent KIE of 3.2 ± 0.1. Quantum chemical matching of the isotope effects supports a concerted, loose, highly asymmetric DNAN transition state with a Pauling bond order of 0.17 to the attacking hydroxide oxygen nucleophile and 0.53 to the departing deoxyadenosine. The reaction coordinate distance is 4.7 Å from attacking the hydroxyl oxygen to departing 5′-deoxyadenosine oxygen. The solvent KIE is consistent with a near-midpoint proton transfer from the His215 catalytic site proton donor to the deoxyadenosine 5′-oxygen in the transition state. This is the first triphosphohydrolase transition state to be characterized and the first use of a 33P primary isotope effect to characterize a phosphotransferase transition state.
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