Human 2'-Deoxynucleoside 5'-Phosphate N -Hydrolase 1: Mechanism of 2'-Deoxyuridine 5'-Monophosphate Hydrolysis.
Suneeta DeviAnna E CarberryGreice M ZickuhrAlison L DicksonDavid J HarrisonRafael G da SilvaPublished in: Biochemistry (2023)
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 p K a of 6.4 must be deprotonated, while two groups with an average p K 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.
Keyphrases
- mass spectrometry
- liquid chromatography
- endothelial cells
- induced pluripotent stem cells
- anaerobic digestion
- ionic liquid
- high resolution
- pluripotent stem cells
- high resolution mass spectrometry
- gas chromatography
- high throughput
- simultaneous determination
- computed tomography
- ms ms
- high performance liquid chromatography
- dna binding
- machine learning
- solar cells
- contrast enhanced