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Publication Abstract from PubMed

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 k(cat)/K(M), two groups with an average pK(a) of 6.4 must be deprotonated, while two groups with an average pK(a) 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 k(cat)/K(M). Solvent deuterium isotope effects on k(cat)/K(M) and k(cat) were inverse and unity, respectively. A reaction mechanism for dUMP hydrolysis is proposed.

Human 2'-Deoxynucleoside 5'-Phosphate N-Hydrolase 1: Mechanism of 2'-Deoxyuridine 5'-Monophosphate Hydrolysis.,Devi S, Carberry AE, Zickuhr GM, Dickson AL, Harrison DJ, da Silva RG Biochemistry. 2023 Aug 15. doi: 10.1021/acs.biochem.3c00369. PMID:37582341^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.