Human 2'-deoxynucleoside 5'-phosphate N-hydrolase 1: the catalytic roles of Tyr24 and Asp80

Anna Ellen Carberry, Suneeta Devi, David James Harrison, R.G. da Silva*

*Corresponding author for this work

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Abstract

The human enzyme 2′-deoxynucleoside 5′-phosphate N-hydrolase 1 (HsDNPH1) catalyses the hydrolysis of 5-hydroxymethyl-2′-deoxyuridine 5′-phosphate to generate 5-hydroxymethyluracil and 2-deoxyribose-5-phosphate via a covalent 5-phospho-2-deoxyribosylated enzyme intermediate. HsDNPH1 is a promising target for inhibitor development towards anticancer drugs. Here, site-directed mutagenesis of conserved active-site residues, followed by HPLC analysis of the reaction and steady-state kinetics are employed to reveal the importance of each of these residues in catalysis, and the reaction pH-dependence is perturbed by each mutation. Solvent deuterium isotope effects indicate no rate-limiting proton transfers. Crystal structures of D80N-HsDNPH1 in unliganded and substrate-bound states, and of unliganded D80A- and Y24F-HsDNPH1 offer atomic level insights into substrate binding and catalysis. The results reveal a network of hydrogen bonds involving the substrate and the E104-Y24-D80 catalytic triad and are consistent with a proposed mechanism whereby D80 is important for substrate positioning, for helping modulate E104 nucleophilicity, and as the general acid in the first half-reaction. Y24 positions E104 for catalysis and prevents a catalytically disruptive close contact between E104 and D80.
Original languageEnglish
Article numbere202400047
Number of pages10
JournalChemBioChem
Volume25
Issue number7
Early online date29 Feb 2024
DOIs
Publication statusPublished - 2 Apr 2024

Keywords

  • DNPH1
  • pH-Rate profiles
  • Biocatalysis
  • Cancer
  • Isotope effects

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