Human 2'-deoxynucleoside 5'-phosphate N-hydrolase 1: mechanism of 2'-deoxyuridine 5'-monophosphate hydrolysis

The enzyme 2′-deoxynucleoside 5′-phosphate N-hydrolase 1 (DNPH1) catalyzes the N-ribosidic bond cleavage of 5-hydroxymethyl-2′-deoxyuridine 5′-monophosphate to generate 2-deoxyribose 5-phosphate and 5-hydroxymethyluracil. DNPH₁ accepts other 2′-deoxynucleoside 5′-monophosphates as slow-reacting substrates. DNPH₁ inhibition is a promising strategy to overcome resistance to and potentiate anticancer poly(ADP-ribose) polymerase inhibitors. We solved the crystal structure of unliganded human DNPH₁ and took advantage of the slow reactivity of 2′-deoxyuridine 5′-monophosphate (dUMP) as a substrate to obtain a crystal structure of the DNPH₁:dUMP Michaelis complex. In both structures, the carboxylate group of the catalytic Glu residue, proposed to act as a nucleophile in covalent catalysis, forms an apparent low-barrier hydrogen bond with the hydroxyl group of a conserved Tyr residue. The crystal structures are supported by functional data, with liquid chromatography–mass spectrometry analysis showing that DNPH₁ incubation with dUMP leads to slow yet complete hydrolysis of the substrate. A direct UV-vis absorbance-based assay allowed characterization of DNPH₁ kinetics at low dUMP concentrations. A bell-shaped pH-rate profile indicated that acid–base catalysis is operational and that for maximum kcat/KM, two groups with an average pKa of 6.4 must be deprotonated, while two groups with an average pKa of 8.2 must be protonated. A modestly inverse solvent viscosity effect rules out diffusional processes involved in dUMP binding to and possibly uracil release from the enzyme as rate limiting to kcat/KM. Solvent deuterium isotope effects on kcat/KM and kcat were inverse and unity, respectively. A reaction mechanism for dUMP hydrolysis is proposed.