Influence of effectors on the rate of reaction of reduced ribonucleoside triphosphate reductase with N-ethylmaleimide

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Ribonucleoside triphosphate reductase from Lactobacillus leichmannii, after reduction by exposure to dithiothreitol, has been alkylated with N-ethylmaleimide. Under conditions where the unreduced enzyme does not incorporate N-ethylmaleimide residues, the reduced enzyme is rapidly alkylated to the extent of one N-ethylmaleimide per molecule of enzyme. Loss of enzyme activity parallels the incorporation of N-ethylmaleimide. The value of the second-order rate constant for the alkylation at 0 °C of the reduced enzyme is influenced by the presence of some of the effectors of the enzyme, e.g., dATP at 200 μ m reduces this parameter from 0.61 to 0.33 m m −1 min −1. The addition of coenzyme B 12 did not significantly affect the rate of alkylation of the reduced enzyme nor did it change the rate of alkylation of the dATP-reduced enzyme complex. Reduced enzyme, freed of dithiol, was shown to be unable to convert CTP stoichiometrically to dCTP when all of the usual enzyme assay components, except the dithiol, were present, nor did addition of CTP to the otherwise complete mixture decrease the level of N-ethylmaleimide-reactive thiol. However, the subsequent addition of dithiol was found to result in essentially complete reduction of CTP to dCTP. Hence, although reduction of the enzyme is probably required to generate an active form of the enzyme, the reduced enzyme does not appear to be capable of transferring its reducing equivalents stoichiometrically to the substrate to form dCTP from CTP. These results are discussed in terms of the mechanism of action of this enzyme.