Human 2'-deoxynucleoside 5'-phosphate N-hydrolase 1 : the catalytic roles of Tyr24 and Asp80
Date
02/04/2024Metadata
Show full item recordAbstract
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.
Citation
Carberry , A E , Devi , S , Harrison , D J & da Silva , R G 2024 , ' Human 2'-deoxynucleoside 5'-phosphate N-hydrolase 1 : the catalytic roles of Tyr24 and Asp80 ' , ChemBioChem , vol. 25 , no. 7 , e202400047 . https://doi.org/10.1002/cbic.202400047
Publication
ChemBioChem
Status
Peer reviewed
ISSN
1439-4227Type
Journal article
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