Studies on the catalysis of carbon-cobalt bond homolysis by ribonucleosidetriphosphate reductase: Evidence for concerted carbon-cobalt bond homolysis and thiyl radical formation

Ribonucleotide reductases (RNRs) catalyze the rate-determining step in DNA biosynthesis: conversion of nucleotides to deoxynucleotides. The RNR from L actobacillus leichmannii utilizes adenosylcobalamin (AdoCbl) as a cofactor and, in addition to nucleotide reduction, catalyzes the exchange of tritium from [5'-H-3]-AdoCbl with solvent. Examination of this exchange reaction o ffers a unique opportunity to investigate the early stages in the nucleotid e reduction process [Licht S. S., Gerfen, G. J., and Stubbe, J. (1996) Scie nce 271, 477-481]. The kinetics of and requirements for this exchange react ion have been examined in detail. The turnover number for H-3 washout is 0. 3 s(-1), and it requires an allosteric effector dGTP (K-m = 17 +/- 3 mu M), AdoCbl (K-m = 60 +/- 9 mu M) and no external reductant. The effects of act ive-site mutants of RTPR (C119S, C419S, C731S, C736S, and C408S) on the rat e of the exchange reaction have been determined, and only C408 is essential for this process. The exchange reaction has previously been monitored by s topped-flow UV-vis spectroscopy, and cob(II)alamin was shown to be formed w ith a rate constant of 40 s(-1) [Tamao, Y., and Blakley, R. L. (1973) Bioch emistry 12, 24-34]. This rate constant has now been measured in D2O, with [ 5'-H-2(2)]-AdoCbl in H2O, and with [5'-H-2(2)]-AdoCbl in D2O. A comparison of these results with those for AdoCbl in H2O revealed k(H)/k(D) Of 1.6, 1. 7, and 2.7, respectively. The absolute amounts of cob(II)alamin generated w ith [5'-H-2(2)]-AdoCbl in D2O in comparison with AdoCbl in H2O reveal twice as much cob(II)alamin in the former case. Similar transient kinetic studie s with C408S RTPR reveal no cob(II)alamin formation. These experiments allo w proposal of a minimal mechanism for this exchange reaction in which RNR c atalyzes homolysis of the carbon-cobalt bond in a concerted fashion, to gen erate a thiyl radical on C408, cob(II)alamin, and 5'-deoxyadenosine.