In vitro regulation of ribonucleotide reductase from Rhizobium meliloti



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Ribonucleotide reductase from Rhizobium meliloti was purified 8- to 10-fold, yielding a specific activity of 3250 nmoles dGDP formed/mg protein/hr. The optimum concentrations of dihydrolipoate, B[lowered 12] coenzyme, GDP and GTP were 20 mM, 30 uM, 0.2 mM and 1.6 mM, respectively. Although Mg[raised ++] and ATP were not required for enzyme activity as has been reported in other systems which reduce ribonucleoside diphosphates, these compounds did under certain conditions effect the rate of ribonucleotide reduction. In general, the addition of Mg[raised ++] at optimum GDP or GTP concentrations did not effect ribonucleotide reductase activity, whereas the addition of ATP was inhibitory. At one-half optimum substrate concentrations Mg[raised ++] and Mg[raised ++] plus ATP stimulated GDP and GTP reduction. When Ca[raised ++] was substituted for Mg[raised ++] essentially no effect was observed on reductase activity at optimum substrate concentrations; the substitution of Mn[raised ++] for Mg[raised ++] inhibited enzyme activity. At one-half optimum substrate concentrations, Ca[raised ++] stimulated enzyme activity, while Mn[raised ++] inhibited reductase activity slightly.