Human 2 ´-Deoxynucleoside 5´-Phosphate N-Hydrolase 1: The Catalytic Roles of Tyr24 and Asp80

Chembiochem. 2024 Feb 13:e202400047. doi: 10.1002/cbic.202400047. Online ahead of print.ABSTRACTThe 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.PMID:38350003 | DOI:10.1002/cbic.202400047

Source: Chembiochem - February 13, 2024 Category: Biochemistry Authors: Anna E Carberry Suneeta Devi David J Harrison Rafael G da Silva Source Type: research