Purification and Properties of Cobamide-dependent Ribonucleotide Reductase from Lactobacillus leichmannii

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Abstract A procedure has been described for the purification of ribonucleotide reductase from Lactobacillus leichmannii, a rich source of enzyme. The enzyme is monodisperse in the ultracentrifuge and on cellulose acetate electrophoresis. Only trace impurities appear on polyacrylamide gel electrophoresis, but this procedure, performed at pH 9.5, yields two major bands. This may reflect alteration of the enzyme by the conditions of electrophoresis. The sedimentation coefficient is 5.8 S, and the molecular weight calculated from sedimentation equilibrium analysis is approximately 110,000. The preferred substrates are ribonucleoside triphosphates. Purified enzyme requires 5,6-dimethylbenzimidazolylcobamide coenzyme and dihydrolipoate or the thioredoxinthioredoxin reductase system from Escherichia coli B. The requirements for divalent cations and adenosine triphosphate vary, depending upon the presence of one another and the identity of the substrate. For example, in the absence of Mg++, the ATP requirement for cytidine triphosphate reduction is decreased, and, in the absence of ATP, Mg++ inhibits CTP, uridine triphosphate, and guanosine triphosphate reduction. It is concluded that divalent cations function in regulating the rates at which different substrates are reduced. Studies of the role of ATP in CTP reduction show that dATP can replace ATP. ATP and dATP both participate catalytically and decrease the apparent Km for CTP. Evidence is presented which suggests that ATP may be converted to dATP, which then serves as an allosteric effector for CTP reduction.