Allosteric transitions associated with the binding of substrate and effector ligands to T2 phage induced deoxycytidylate deaminase.
The binding characteristics of T2 phage induced deoxycytidylate deaminase were examined through the use of ultrafiltration and equilibrium dialysis. The positive effectors, 5-(hydroxymethyl)deoxycytidine 5'-triphosphate and deoxycytidine 5'-triphosphate, were bound in a highly cooperative manner, which is consistent with the allosteric effects promoted by these compounds. Their respective S0.5 values were 8 and 2 microM. A similar degree of cooperativity was associated with the binding of such competitive inhibitors of deoxycytidylate deaminase as dGMP, 4-N-hydroxydeoxycytidine 5'-monophosphate, and tetrahydrodeoxyuridylate. The negative effector, dTTP, also inhibited the binding of dCTP in a pH-dependent manner, which is consistent with its previously demonstrated inhibition of catalysis [Maley, G. F., Guarino, D. U., & Maley, F. (1972) J. Biol. Chem. 247, 931-939]. The binding of dTTP could be demonstrated only at low phosphate concentrations and did not appear to be cooperative. The number of binding sites for the allosteric ligands, substrate, and substrate inhibitors was shown to be six, which coincides with the number of enzyme subunits. It was established by CD difference spectroscopy that dCTP, at concentrations normally employed to demonstrate enzyme activation, effects a dramatic conformation transition in the deaminase, as indicated by a sharp decrease in ellipticity at about 280 nm. The nature of this response suggests that the microenvironment of some of the enzyme's tyrosyl residues had been perturbed by the presence of this allosteric nucleotide.
D002942:Circular Dichroism; D003844:DCMP Deaminase / Q000096:biosynthesis / Q000378:metabolism*; D003845:Deoxycytosine Nucleotides / Q000378:metabolism; D004790:Enzyme Induction; D007700:Kinetics; D008433:Mathematics; D009709:Nucleotide Deaminases / Q000378:metabolism*; D013379:Substrate Specificity; D013604:T-Phages / Q000201:enzymology*; D013942:Thymine Nucleotides / Q000494:pharmacology
D003845:Deoxycytosine Nucleotides; D013942:Thymine Nucleotides; C024107:2'-deoxycytidine 5'-triphosphate; D009709:Nucleotide Deaminases; D003844:DCMP Deaminase; C024157:thymidine 5'-triphosphate