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 composition to the T2-phage-induced deaminase. Neither enzyme is active in the absence of dCTP or its natural activator, 5-hydroxymethyl-dCTP. However, on changing the arginine (Arg) at residue 115 of the T4-deaminase to either 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 compared on the basis of specific activity, however, the mutants are about 40-50% of the wild-type (WT)-enzyme's specific activity. Molecular weight analysis on the wild-type and mutant deaminases using HPLC size exclusion chromatography revealed that the wild-type deaminase was basically a hexamer, particularly in the presence of dCTP, regardless of the extent of dilution. Under similar conditions, R115E remained a dimer, whereas R115Q and F112A varied from hexamers to dimers particularly at concentrations normally present in the assay solution. Activity measurements appear to support the conclusion 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 activities with the WT deaminase only marginally restored.
Keefe
R G
RG
Wadsworth Center, New York State Department of Health, Albany, New York 12201-0509, USA.
Maley
G F
GF
Saxl
R L
RL
Maley
F
F
D001120:Arginine / Q000737:chemistry; D017122:Bacteriophage T4 / Q000201:enzymology* / Q000502:physiology*; D001665:Binding Sites; D002850:Chromatography, Gel; D003844:DCMP Deaminase / Q000235:genetics* / Q000378:metabolism; D003845:Deoxycytosine Nucleotides / Q000502:physiology*; D004492:Edetic Acid / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry; D005973:Glutamine / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011489:Protein Denaturation; D020673:Protein Renaturation; D017433:Protein Structure, Secondary; D013997:Time Factors; D015032:Zinc / Q000737:chemistry
D003845:Deoxycytosine Nucleotides; D005973:Glutamine; C024107:2'-deoxycytidine 5'-triphosphate; D018698:Glutamic Acid; D001120:Arginine; D004492:Edetic Acid; D003844:DCMP Deaminase; D015032:Zinc