T4 phage ribonucleotide reductase. Allosteric regulation in vivo by thymidine triphosphate.
Based upon analyses of purified enzyme preparations, T4 bacteriophage-coded ribonucleotide reductase is considered to be relatively insensitive to control by allosteric inhibition. However, two factors suggest that CDP reduction to dCDP is feedback-controlled by dTTP in infected cells. First, the pool of 5-hydroxymethyldeoxycytidine triphosphate, which expands manyfold upon infection by a dCMP deaminase-deficient T4 mutant, shrinks to near-normal levels as a consequence of dTTP accumulation, and ribonucleotide reductase is the only apparent control point. Second, analysis of mutagenesis by 5-bromodeoxyuridine suggests that most induced mutations result from localized pool depletion of 5-hydroxymethyl-dCTP at replication sites, as if 5-bromo-dUTP were behaving like dTTP in inhibiting the CDP reductase activity of the phage enzyme. We found that CDP reductase activity in crude extracts of T4 phage-infected bacteria is sensitive to inhibition by either dTTP or 5-bromo-dUTP, at concentrations as low as 0.01 mM. However, in partially purified enzyme preparations that sensitivity is lost. Although we don't know the basis for this loss of feedback sensitivity, the results suggest that kinetic properties of enzymes in intact cells are determined by the cellular milieu in ways not apparent from analysis of purified enzymes.
Ji
J P
JP
Department of Biochemistry and Biophysics, Oregon State University, Corvallis 97331-6503.
Sargent
R G
RG
Mathews
C K
CK
D000494:Allosteric Regulation; D001483:Base Sequence; D003565:Cytidine Diphosphate / Q000378:metabolism; D004247:DNA; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009711:Nucleotides / Q000378:metabolism; D010084:Oxidation-Reduction; D012264:Ribonucleotide Reductases / Q000378:metabolism*; D013604:T-Phages / Q000201:enzymology*; D013942:Thymine Nucleotides / Q000378:metabolism*
D009711:Nucleotides; D013942:Thymine Nucleotides; D003565:Cytidine Diphosphate; D004247:DNA; D012264:Ribonucleotide Reductases; C024157:thymidine 5'-triphosphate