A T4-phage deoxycytidylate deaminase mutant that no longer requires deoxycytidine 5 '-triphosphate for activation

A deoxycytidylate (dCMP) deaminase encoded in T4-bacteriophage DNA that is induced on phage infection of Escherichia coli was shown earlier (Maley, G. F., Duceman, B. W., Wang, A. M., Martinez, J. M., and Maley, F. (1990) J. Biol. Chem. 265, 47-51) to be similar in size, properties, and amino acid c omposition to the T2-phage-induced deaminase. Neither enzyme is active in t he absence of dCTP or its natural activator, 5-hydroxymethyl-dCTP. However, on changing the arginine (Arg) at residue 115 of the T4-deaminase to eithe r a glutamate (R115E) or a glutamine (R115Q), the resulting mutant enzymes were active in the absence of dCTP, with each mutant possessing a turnover number or k(cat) that is about 15% that of the wild-type deaminase, When co mpared on the basis of specific activity, however, the mutants are about 40 -50% of the wild-type (WT)-enzyme's specific activity. Molecular weight ana lysis on the wildtype and mutant deaminases using HPLC size exclusion chrom atography revealed that the wild-type deaminase was basically a hexamer, pa rticularly in the presence of dCTP, regardless of the extent of dilution. U nder similar conditions, R115E remained a dimer, whereas R115Q and F112A va ried from hexamers to dimers particularly at concentrations normally presen t in the assay solution. Activity measurements appear to support the conclu sion that the hexameric form of the enzyme is activated by dCTP, while the dimer is not. Another feature emphasizing the difference between the WT and mutant deaminases was observed on their denaturation-renaturation in EDTA, which revealed the mutants to be restored to 50% of their original activit ies with the WT deaminase only marginally restored.