Studies on the allosteric regulation of ribonucleotide reductase of Rhizobium meliloti



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Ribonucleotide reductase was purified from Rhizobium meliloti, Strain F-28, by ammonium sulfate fractionation and chromatography on Sephadex G-200 and DEAE-cellulose. The enzyme was purified at least 25-fold by these procedures. The purified enzyme was capable of reducing the 5'-diphosphates of guanosine, cytidine, and adenosine (GDP, CDP, and ADP). Reduction of uridine diphosphates (UDP) could not be clearly established by the assay procedure. The allosteric regulation of the reduction of the three ribonucleoside diphosphate substrates by deoxyribonucleoside triphosphates was determined using the purified enzyme. The rate of GDP reduction was decreased by all four deoxyribonucleoside triphosphates at concentrations of 1 X 10[raised -6] M or greater. Deoxy-GTP and dTTP were the most effective negative effectors reducing GDP reduction by 80 and 60% at concentrations of 5 X 10[raised -4] M and 5 X 10[raised -5] M, respectively. ADP reduction was stimulated 300% by dGTP at 1 X 10[raised -6]M. Deoxy-ATP and dTTP however were negative effectors. The rate of CDP reduction was stimulated 300% by dATP at 5 X 10[raised -6] M. Deoxy-TTP at low concentrations (5 X 10[raised -8] M) stimulated CDP reduction 180%. Deoxy-GTP at 1 X 10[raised -5] M was a negative effector, while dCTP had no influence on CDP reduction. A suggested pattern for the in vivo ribonucleotide reduction based on the results of this study would be as follows: the initial conversion of GDP to dGDP which requires no positive effectors. Deoxy-GTP then stimulates the reduction of ADP to dADP. Deoxy-ATP in turn stimulates the formation of dCDP from CDP. Although it has not been demonstrated at this time UDP reduction should be stimulated by dCTP. A universal negative inhibitor was not found. Ribonucleotide diphosphate reductase was inhibited by Mg[raised ++] at concentrations of 1 to 32 mM. This was shown not to be the result of Mg[raised ++] precipitation of dihydrolipoate from the reaction mixtures as was expected.