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

Abstract The human enzyme 2′‐deoxynucleoside 5′‐phosphate N‐ hydrolase 1 ( Hs DNPH1) 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. Hs DNPH1 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‐ Hs DNPH1 in unliganded and substrate‐bound states, and of unliganded D80A‐ and Y24F‐ Hs DNPH1 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.