Ribonucleotide reductases (RNRs) catalyze the rate-determining step in DNA biosynthesis: conversion of nucleotides to deoxynucleotides. The RNR from Lactobacillus leichmannii utilizes adenosylcobalamin (AdoCbl) as a cofactor and, in addition to nucleotide reduction, catalyzes the exchange of tritium from [5'-3H]-AdoCbl with solvent. Examination of this exchange reaction offers a unique opportunity to investigate the early stages in the nucleotide reduction process [Licht S.S., Gerfen, G. J., and Stubbe, J. (1996) Science 271,477-481]. The kinetics of and requirements for this exchange reaction have been examined in detail. The turnover number for 3H washout is 0.3 s- 1, and it requires an allosteric effector dGTP (K(m) = 17 ± 3 μM), AdoCbl (K(m) = 60 ± 9 μM) and no external reductant. The effects of active-site mutants of RTPR (C119S, C419S, C731S, C736S, and C408S) on the rate of the exchange reaction have been determined, and only C408 is essential for this process. The exchange reaction has previously been monitored by stopped-flow UV-vis spectroscopy, and cob(II)alamin was shown to be formed with a rate constant of 40 s-1 [Tamao, Y., and Blakley R. L. (1973) Biochemistry 12, 24-34]. This rate constant has now been measured in D2O, with [5'-2H2]- AdoCbl in H2O, and with [5'-2H2]-AdoCbl in D2O. A comparison of these results with those for AdoCbl in H2O revealed k(H)/(D) of 1.6, 1.7, and 2.7, respectively. The absolute amounts of cob(II)alamin generated with [5'2H2]- AdoCbl in D2O in comparison with AdoCbl in H2O reveal twice as much cob(II)alamin in the former case. Similar transient kinetic studies with C408S RTPR reveal no cob(II)alamin formation. These experiments allow proposal of a minimal mechanism for this exchange reaction in which RNR catalyzes homolysis of the carbon-cobalt bond in a concerted fashion, to generate a thiyl radical on C408, cob(II)alamin, and 5'-deoxyadenosine.
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