Biomaterial Database

Studies on the catalysis of carbon-cobalt bond homolysis by ribonucleoside triphosphate reductase: evidence for concerted carbon-cobalt bond homolysis and thiyl radical formation. 1999

S S Licht, and S Booker, and J Stubbe

Departments of Chemistry and Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

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 (Km = 17 +/- 3 microM), AdoCbl (Km = 60 +/- 9 microM) 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 kH/kD 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.

UI	MeSH Term	Description	Entries
D007553	Isotope Labeling	Techniques for labeling a substance with a stable or radioactive isotope. It is not used for articles involving labeled substances unless the methods of labeling are substantively discussed. Tracers that may be labeled include chemical substances, cells, or microorganisms.	Isotope Labeling, Stable,Isotope-Coded Affinity Tagging,Isotopically-Coded Affinity Tagging,Affinity Tagging, Isotope-Coded,Affinity Tagging, Isotopically-Coded,Isotope Coded Affinity Tagging,Labeling, Isotope,Labeling, Stable Isotope,Stable Isotope Labeling,Tagging, Isotope-Coded Affinity,Tagging, Isotopically-Coded Affinity
D007700	Kinetics	The rate dynamics in chemical or physical systems.
D007778	Lactobacillus	A genus of gram-positive, microaerophilic, rod-shaped bacteria occurring widely in nature. Its species are also part of the many normal flora of the mouth, intestinal tract, and vagina of many mammals, including humans. Lactobacillus species are homofermentative and ferment a broad spectrum of carbohydrates often host-adapted but do not ferment PENTOSES. Most members were previously assigned to the Lactobacillus delbrueckii group. Pathogenicity from this genus is rare.
D010084	Oxidation-Reduction	A chemical reaction in which an electron is transferred from one molecule to another. The electron-donating molecule is the reducing agent or reductant; the electron-accepting molecule is the oxidizing agent or oxidant. Reducing and oxidizing agents function as conjugate reductant-oxidant pairs or redox pairs (Lehninger, Principles of Biochemistry, 1982, p471).	Redox,Oxidation Reduction
D011994	Recombinant Proteins	Proteins prepared by recombinant DNA technology.	Biosynthetic Protein,Biosynthetic Proteins,DNA Recombinant Proteins,Recombinant Protein,Proteins, Biosynthetic,Proteins, Recombinant DNA,DNA Proteins, Recombinant,Protein, Biosynthetic,Protein, Recombinant,Proteins, DNA Recombinant,Proteins, Recombinant,Recombinant DNA Proteins,Recombinant Proteins, DNA
D002384	Catalysis	The facilitation of a chemical reaction by material (catalyst) that is not consumed by the reaction.	Catalyses
D003038	Cobamides		Deoxyadenosinecobalamins,Vitamin B 12 Coenzymes,Vitamin B12 Coenzymes,B12 Coenzymes, Vitamin,Coenzymes, Vitamin B12
D012264	Ribonucleotide Reductases		Ribonucleotide Reductase,Reductase, Ribonucleotide,Reductases, Ribonucleotide
D014316	Tritium	The radioactive isotope of hydrogen also known as hydrogen-3. It contains two NEUTRONS and one PROTON in its nucleus and decays to produce low energy BETA PARTICLES.	Hydrogen-3,Hydrogen 3
D016297	Mutagenesis, Site-Directed	Genetically engineered MUTAGENESIS at a specific site in the DNA molecule that introduces a base substitution, or an insertion or deletion.	Mutagenesis, Oligonucleotide-Directed,Mutagenesis, Site-Specific,Oligonucleotide-Directed Mutagenesis,Site-Directed Mutagenesis,Site-Specific Mutagenesis,Mutageneses, Oligonucleotide-Directed,Mutageneses, Site-Directed,Mutageneses, Site-Specific,Mutagenesis, Oligonucleotide Directed,Mutagenesis, Site Directed,Mutagenesis, Site Specific,Oligonucleotide Directed Mutagenesis,Oligonucleotide-Directed Mutageneses,Site Directed Mutagenesis,Site Specific Mutagenesis,Site-Directed Mutageneses,Site-Specific Mutageneses