Mechanistic studies of ribonucleoside triphosphate reductase from Lactobacillus leichmannii
The mechanism of action of the adenosylcobalamin (AdoCbl)-dependent ribonucleoside triphosphate reductase (RTPR) was investigated using isotope effect and substrate specificity studies. These experiments were conducted on RTPR purified by a new method from Lactobacillus leichmannii. Isotope effect studies using (3{prime}-{sup 3}H)UTP and (3{prime}-{sup 3}H)ATP demonstrated that the 3{prime} C-H bond of the nucleotide is cleaved in order to cleave the 2{prime} C-OH bond. AdoCbl does not act as a direct H abstractor from the 3{prime} position of the substrate, but instead is thought to act as a radical chain initiator to generate an amino acid radical on the enzyme. Further support for this enzyme mediated cleavage of the 3{prime} C-H bond of the nucleotide and the novel role of AdoCbl came from studies using (3{prime}{sup 3}H)2{prime}-chloro-2{prime}-deoxyuridine 5{prime}-triphosphate ((3{prime}-{sup 3}H)CIUTP). Evidence is presented that during the course of this reaction, the {sup 3}H abstracted from the 3{prime} position of (3{prime}-{sup 3}H)CIUTP was either exchanged with the solvent or returned to the {beta} face of the 2{prime} position to produce (2{prime}{sup 3}H)-2{prime}-deoxy-3{prime}-ketoUTP. This result demonstrates that RTPR is capable of catalyzing a rearrangement reaction. The significance of the RTPR-catalyzed rearrangement with respect to the AdoCbl-dependent enzymes which catalyze rearrangements is discussed.
- Research Organization:
- Wisconsin Univ., Madison, WI (USA)
- OSTI ID:
- 7117846
- Resource Relation:
- Other Information: Thesis (Ph. D.)
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
LACTOBACILLUS
ENZYME ACTIVITY
OXIDOREDUCTASES
BIOCHEMICAL REACTION KINETICS
ISOTOPE EFFECTS
AMINO ACIDS
PURIFICATION
SUBSTRATES
TRITIUM COMPOUNDS
BACTERIA
CARBOXYLIC ACIDS
ENZYMES
HYDROGEN COMPOUNDS
KINETICS
MICROORGANISMS
ORGANIC ACIDS
ORGANIC COMPOUNDS
REACTION KINETICS
550201* - Biochemistry- Tracer Techniques