An enzyme-coupled microplate assay for activity and inhibition of hmdUMP hydrolysis by DNPH1

Human 2′-Deoxynucleoside 5′-Phosphate N-Hydrolase 1: Mechanism of 2′-Deoxyuridine 5′-Monophosphate Hydrolysis

“…Fitting the data in H 2 O to eq yielded a K M of 8 ± 1 mM, in excellent agreement with that previously reported, and a k cat of 3.0 ± 0.2 min –1 , fourfold lower than a previously published value . For comparison, a K M of 6.3 μM and a k cat of 19.8 min –1 have been recently reported for Hs DNPH1-catalyzed hydrolysis of the physiological substrate 5hmdUMP at 37 °C . The data in D 2 O were best fitted to eq , which excludes an isotope effect on , resulting in a of 0.62 ± 0.07.…”

“…6 For comparison, a K M of 6.3 μM and a k cat of 19.8 min −1 have been recently reported for HsDNPH1-catalyzed hydrolysis of the physiological substrate 5hmdUMP at 37 °C. 48 The data in D 2 O were best fitted to eq 5, which excludes an isotope effect on rules out a rate-limiting proton-transfer step from the HsDNPH1:dUMP complex to release of 2deoxyribose 5-phosphate. 49 Proposed Catalytic Mechanism.…”

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

“…[6] Owing to the relatively low sensitivity of our assay, the lowest concentration of 5hmdUMP (2.5 μM) that resulted in a reliable rate measurement was only marginally below the Michaelis constant (K M ) of 3.5 � 0.4 μM; thus, this value should be interpreted with caution, even though it lies within 2-fold of that recently reported. [6] To permit comparison with recently reported kinetic parameters for WT and mutant HsDNPH1, [7] all subsequent kinetic measurements were carried out at 25 °C. To probe the role of acid-base catalysis in the HsDNPH1 hydrolysis of 5hmdUMP, a pH-rate profile was obtained.…”

“…Kinetics and protein denaturation data were analysed by the non-linear regression function of SigmaPlot 13.0 (SPSS Inc.). Substrate saturation curves were fitted to equation (1); bell-shaped pH-rate profiles were fitted to equation (2); thermal melting data, to equation (3); the reaction activation energies were calculated with equation (4); pH-rate profiles with pK a either on acidic limb only or on basic limb only were fitted to either equation ( 5) or (6), respectively; substrate saturation curves where pseudo-first order reaction could not be assumed were fitted to equation (7); substrate-dependence of the initial rate at low subtrate concentrations was fitted to equation (8). In equations 1, 2, and 4-8, v is the initial rate, k cat is the steady-state turnover number, K M is the Michaelis constant, E T is total enzyme concentration, S is the concentration of substrate, C is the pHindependent value of k cat , H is the proton concentration, K a1 and K a2 are apparent acid dissociation constants, h is Planck's constant, k B is Boltzmann constant, R is the ideal gas constant, T is the absolute temperature, ΔG � is the reaction activation energy, k is the transmission coefficient (assumed to be 1).…”

“…[5] HsDNPH1's specificity constant k cat /K M is 1,940-fold higher for 5hmdUMP than for dUMP at 37 °C. [4,6] Structural and kinetic studies using dUMP as substrate hypothesized a doubledisplacement mechanism where the conserved E104 residue would act as a nucleophile to form a covalent 5-phospho-2deoxyribosyl-enzyme intermediate in the first half-reaction, and the conserved D80 residue would serve as the general base to activate a water molecule to hydrolyse this intermediate in the second half-reaction. [4] Intriguingly, E104 was found in crystallo in a strong hydrogen bond (H-bond) with the conserved residue Y24.…”