Abstract
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.
Highlights
1 5-Hydroxymethyl-dCTP is the most probable activator of the T4phage deaminase, since it is incorporated into the phage DNA in place of dCTP
In enzyme assays where dCTP was absent from the assay solution, activity was observed immediately upon the addition of R115E or R115Q, as shown earlier for F112A [10], whereas wild-type enzyme activity could not be detected until dCTP was added to the reaction cuvette
Since our initial observation that dCMP deaminase, at least in eukaryotes, is an allosteric enzyme activated by dCTP and inhibited by dTTP [13], a major objective has been to determine how these effectors manage to regulate this enzyme
Summary
Chemicals and Reagents—High purity dCMP, dCTP, and dTTP were purchased from Sigma. Other chemicals and reagents used for bacterial cell growth, buffers, enzyme purification and assay, and polyacrylamide electrophoresis were of reagent grade and purchased from the following commercial sources: Sigma, Fisher, Mallinckrodt Baker, Inc., (Paris, KY), or Life Technologies, Inc. The assay solution in which deaminase activity was measured contained 10 mM Tris-HCl, pH 8.0, 0.5 mM dCMP, 0.5 mM MgCl2, 5 mM 2-mercaptoethanol, with or without 20 M dCTP, depending on the enzyme to be assayed. Due to the relatively high turnover of the WT deaminase (ϳ2.5 ϫ 104 mol of dCMP deaminated minϪ1 molϪ1 hexamer), enzyme samples were routinely diluted to 15–35 g/ml protein with a solution of 0.2 M potassium phosphate, pH 7.5, 10% ethylene glycol, 0.1 M 2-mercaptoethanol, and 20 M dCTP, from which aliquots were removed for the measurement of enzyme activity. Denaturation-Renaturation Time Course Experiments—One microliter of purified R115E (8.5 g), R115Q (10 g), or wild-type enzyme (3.5 g) was diluted 1:10 in a solution of 0.2 M potassium phosphate, pH 7.5, 6 M guanidine HCl, 0.1 M 2-mercaptoethanol, Ϯ 1.0 mM EDTA. Molecular weight standard proteins (Sigma) used to calibrate the column were: ␣-amylase (200,000), alcohol dehydrogenase (150,000), bovine serum albumin (66,000), carbonic anhydrase (29,000), and horse heart cytochrome c [12,400]
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