Regulation of Cobamide-dependent Ribonucleotide Reductase by Allosteric Effectors and Divalent Cations

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Abstract Factors regulating the rate behavior and substrate specificity of purified cobamide-dependent ribonucleotide reductase of Lactobacillus leichmannii have been examined in detail. Reduction of each of the four common ribonucleoside triphosphates (cytidine triphosphate, uridine triphosphate, adenosine triphosphate, and guanosine triphosphate) is maximally stimulated by a different deoxyribonucleoside triphosphate. These deoxyribonucleotides, conveniently termed effectors, are: deoxyadenosine triphosphate for CTP reduction; deoxycytidine triphosphate for UTP reduction; deoxyguanosine triphosphate for ATP reduction; and thymidine triphosphate for GTP reduction. Only ATP and GTP are actively reduced in the absence of added effector; in both cases, however, reduction is enhanced by prime effector. Deoxyribonucleotides that are not prime effectors for a given reduction may be negative or weakly positive effectors. Effector nucleotides are active at low concentrations (viz. 10-4 m); they are not replaceable by the corresponding diphosphates and monophosphates; and they are in competition with one another for an enzymatic binding site other than the catalytic site. Thus, they are allosteric effectors. Kinetic studies with substrate mixtures suggest the presence of a single catalytic site, but are inconclusive because the reduction product of one substrate (or the substrate itself) may be a positive or negative effector for reduction of the other substrate. Divalent cations (Mg++, Ca++, and Mn++) also have significant stimulatory and inhibitory effects that vary with the substrate, the effector, and the sequence of cation and effector addition. The results indicate that this enzyme, previously shown to be derepressed when folate-free cells are grown in thymine-free medium (and probably repressed by intracellular dTTP), is also subject to a complex network of positive and negative feedback effects in which allosteric effector nucleotides and divalent cations, presumably through modifications of the physical state of the enzyme, determine which ribonucleotides are reduced. It is concluded that these effects may maintain the balance of deoxyribonucleotide synthesis in vivo.