Studies on the catalysis of carbon-cobalt bond homolysis by ribonucleoside triphosphate reductase

Evidence for concerted carbon-cobalt bond homolysis and thiyl radical formation

Stuart S. Licht, Squire J. Booker, JoAnne Stubbe

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)1221-1233
Number of pages13
JournalBiochemistry
Volume38
Issue number4
DOIs
StatePublished - Jan 26 1999

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ribonucleoside-triphosphate reductase
Cobalt
Catalysis
Carbon
Ribonucleotide Reductases
Nucleotides
Lactobacillus leichmannii
Rate constants
Biochemistry
Kinetics
Tritium
cobamamide
Biosynthesis
Reducing Agents
Ultraviolet spectroscopy

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

@article{0de537ff595643bf8ca922a63d451880,
title = "Studies on the catalysis of carbon-cobalt bond homolysis by ribonucleoside triphosphate reductase: Evidence for concerted carbon-cobalt bond homolysis and thiyl radical formation",
abstract = "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.",
author = "Licht, {Stuart S.} and Booker, {Squire J.} and JoAnne Stubbe",
year = "1999",
month = "1",
day = "26",
doi = "10.1021/bi981885i",
language = "English (US)",
volume = "38",
pages = "1221--1233",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
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TY - JOUR

T1 - Studies on the catalysis of carbon-cobalt bond homolysis by ribonucleoside triphosphate reductase

T2 - Evidence for concerted carbon-cobalt bond homolysis and thiyl radical formation

AU - Licht, Stuart S.

AU - Booker, Squire J.

AU - Stubbe, JoAnne

PY - 1999/1/26

Y1 - 1999/1/26

N2 - 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.

AB - 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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U2 - 10.1021/bi981885i

DO - 10.1021/bi981885i

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