Abstract
Human sterile alpha motif and HD-domain-containing protein 1 (SAMHD1) is an allosterically regulated dNTP triphosphohydrolase (dNTP + H(2)O → 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 (18)O and (33)P primary kinetic isotope effects (KIEs) at the α-phosphoryl of the leaving triphosphate group. The intrinsic KIE values for [5'-(18)O]dATP of 1.028 ± 0.003 and for [α-(33)P]dATP of 1.015 ± 0.004 provide insights into the mechanistic details of the SAMHD1 transition state. Solvent (2)H(2)O 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 D(N)A(N) 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 (33)P primary isotope effect to characterize a phosphotransferase transition state.