The energy landscape of N-ribosidic bond cleavage catalysed by 2′-deoxynucleoside 5′-phosphate N-hydrolase 1

The human enzyme 2′-deoxynucleoside 5′-phosphate N-hydrolase 1 (HsDNPH1) catalyses the N-ribosidic bond hydrolysis of the noncanonical nucleotide 5-hydroxymethyl-2′-deoxyuridine 5′-monophosphate (5hmdUMP), producing 5-hydroxymethyluracil and 2-deoxyribose 5-phosphate, preventing 5hmdUMP incorporation into DNA. This reaction is unusual for a nucleoside/nucleotide N-hydrolase as it proceeds by a double-displacement mechanism whereby Glu104 nucleophilically attacks 5hmdUMP to form a covalent 5-phospho-2-deoxyribosylated enzyme intermediate, which is subsequently hydrolysed. Here we used site-directed mutagenesis and UV-VIS differential spectroscopy to show a shift in the 5hmdUMP absorbance spectrum upon binding to HsDNPH1 before N-ribosidic bond cleavage. This spectral shift can be monitored independently in different wavelengths to characterise the kinetics of HsDNPH1-5hmdUMP binary complex formation. The one-step binding mechanism produces a calculated equilibrium dissociation constant in agreement with that obtained by isothermal titration calorimetry. Pre-steady-state kinetics under multiple-turnover conditions revealed absence of a burst of substrate consumption at a wavelength where binding does not lead to change in absorbance. This indicates steps after N-ribosidic bond cleavage are fast. Single-turnover kinetics, where the signal comes solely from the first half-reaction, indicate N-ribosidic bond cleavage in the first half-reaction is determining for kcat. Linear free energy relationships between leaving groups with increased pKa and kcat/KM suggest a late transition state with significant negative charge accumulation in the leaving group during N-ribosidic bond cleavage. These results were complemented by on-enzyme QM/MM calculations of the first half-reaction to reveal an anionic leaving group in an SN2-like transition state with C1′–N1 bond cleavage more advanced than C1–O bond formation with Glu104.