Roles of Cys148 and Asp179 in catalysis by deoxycytidylate hydroxymethylase from bacteriophage T4 examined by site-directed mutagenesis

Site-directed mutagenesis was used to study the roles played by Cys148 and Asp179 in catalysis by deoxycytidylate (dCMP) hydroxymethylase (CH). Results showing that converting Cys148 tp Asp, Gly or Ser causes CH activity to decline 10(super 5)-fold suggests that Cys148 plays anucleophilic role in the process. Asp179 conversion to Asn causes a 1.5 x 10(super 4)-fold decrease in the dCMP kcat/Km value and a 60-fold rise in the dUMP kcat/Km value. Results also suggest that Asp179 stabilizes covalent catalytic intermediates by protonating the pyrimidine-CH adduct's N3.