Inactivation of the Lactobacillus leichmannii ribonucleoside triphosphate reductase by 2'-chloro-2'-deoxyuridine 5'-triphosphate: stoichiometry of inactivation, site of inactivation, and mechanism of the protein chromophore formation

Ashley, G W; Harris, G; Stubbe, J A

doi:10.1021/bi00412a017

Title: Inactivation of the Lactobacillus leichmannii ribonucleoside triphosphate reductase by 2'-chloro-2'-deoxyuridine 5'-triphosphate: stoichiometry of inactivation, site of inactivation, and mechanism of the protein chromophore formation

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Abstract

The ribonucleoside triphosphate reductase (RTPR) of Lactobacillus leichmannii is inactivated by the substrate analogue 2'-chloro-2'-deoxyuridine 5'-triphosphate (ClUTP). Inactivation is due to alkylation by 2-methylene-3(2H)-furanone, a decomposition product of the enzymic product 3'-keto-2'-deoxyuridine triphosphate. The former has been unambiguously identified as 2-((ethylthio)methyl)-3(2H)-furanone, an ethanethiol trapped adduct, which is identical by /sup 1/H NMR spectroscopy with material synthesized chemically. Subsequent to rapid inactivation, a slow process occurs that results in formation of a new protein-associated chromophore absorbing maximally near 320 nm. The terminal stages of the inactivation have now been investigated in detail. The alkylation and inactivation stoichiometries were studied as a function of the ratio of ClUTP to enzyme. The amount of labeling of RTPR increased with increasing ClUTP concentration up to the maximum of approximately 4 labels/RTPR, yet the degree of inactivation did not increase proportionally. This suggests that (1) RTPR may be inactivated by alkylation of a single site and (2) decomposition of 3'-keto-dUTP is not necessarily enzyme catalyzed. The formation of the new protein chromophore was also monitored during inactivation and found to reach its full extent upon the first alkylation . Thus, out of four alkylation sites, only one appears capable of undergoing the subsequent reaction tomore »« less

Authors:: Ashley, G W; Harris, G; Stubbe, J A

Publication Date:: Tue Jun 14 00:00:00 EDT 1988

Research Org.:: Univ. of Wisconsin, Madison (USA)

OSTI Identifier:: 7163842

Resource Type:: Journal Article

Journal Name:: Biochemistry; (United States)

Additional Journal Information:: Journal Volume: 27:12

Country of Publication:: United States

Language:: English

Subject:: 62 RADIOLOGY AND NUCLEAR MEDICINE; 59 BASIC BIOLOGICAL SCIENCES; NUCLEOTIDES; STOICHIOMETRY; OXIDOREDUCTASES; INACTIVATION; NUCLEAR MAGNETIC RESONANCE; ALKYLATION; LACTOBACILLUS; PROTONS; TRITIUM COMPOUNDS; UTP; BACTERIA; BARYONS; CHEMICAL REACTIONS; ELEMENTARY PARTICLES; ENZYMES; FERMIONS; HADRONS; LABELLED COMPOUNDS; MAGNETIC RESONANCE; MICROORGANISMS; NUCLEONS; ORGANIC COMPOUNDS; RESONANCE; 550601* - Medicine- Unsealed Radionuclides in Diagnostics; 550201 - Biochemistry- Tracer Techniques

Citation Formats


                    Ashley, G W, Harris, G, and Stubbe, J A. Inactivation of the Lactobacillus leichmannii ribonucleoside triphosphate reductase by 2'-chloro-2'-deoxyuridine 5'-triphosphate: stoichiometry of inactivation, site of inactivation, and mechanism of the protein chromophore formation.  United States: N. p., 1988. 
        Web.  doi:10.1021/bi00412a017.

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                    Ashley, G W, Harris, G, & Stubbe, J A. Inactivation of the Lactobacillus leichmannii ribonucleoside triphosphate reductase by 2'-chloro-2'-deoxyuridine 5'-triphosphate: stoichiometry of inactivation, site of inactivation, and mechanism of the protein chromophore formation.  United States.  https://doi.org/10.1021/bi00412a017

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                    Ashley, G W, Harris, G, and Stubbe, J A. 1988.  
        "Inactivation of the Lactobacillus leichmannii ribonucleoside triphosphate reductase by 2'-chloro-2'-deoxyuridine 5'-triphosphate: stoichiometry of inactivation, site of inactivation, and mechanism of the protein chromophore formation".  United States.  https://doi.org/10.1021/bi00412a017.

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@article{osti_7163842,

  title        = {Inactivation of the Lactobacillus leichmannii ribonucleoside triphosphate reductase by 2'-chloro-2'-deoxyuridine 5'-triphosphate: stoichiometry of inactivation, site of inactivation, and mechanism of the protein chromophore formation},

  author       = {Ashley, G W and Harris, G and Stubbe, J A},

  abstractNote = {The ribonucleoside triphosphate reductase (RTPR) of Lactobacillus leichmannii is inactivated by the substrate analogue 2'-chloro-2'-deoxyuridine 5'-triphosphate (ClUTP). Inactivation is due to alkylation by 2-methylene-3(2H)-furanone, a decomposition product of the enzymic product 3'-keto-2'-deoxyuridine triphosphate. The former has been unambiguously identified as 2-((ethylthio)methyl)-3(2H)-furanone, an ethanethiol trapped adduct, which is identical by /sup 1/H NMR spectroscopy with material synthesized chemically. Subsequent to rapid inactivation, a slow process occurs that results in formation of a new protein-associated chromophore absorbing maximally near 320 nm. The terminal stages of the inactivation have now been investigated in detail. The alkylation and inactivation stoichiometries were studied as a function of the ratio of ClUTP to enzyme. The amount of labeling of RTPR increased with increasing ClUTP concentration up to the maximum of approximately 4 labels/RTPR, yet the degree of inactivation did not increase proportionally. This suggests that (1) RTPR may be inactivated by alkylation of a single site and (2) decomposition of 3'-keto-dUTP is not necessarily enzyme catalyzed. The formation of the new protein chromophore was also monitored during inactivation and found to reach its full extent upon the first alkylation . Thus, out of four alkylation sites, only one appears capable of undergoing the subsequent reaction to form the new chromophore. Model studies suggest that the new chromophore is due to addition of an amino group to the 5-position of enzyme-bound furanone, followed by ring opening and tautomerization to give a ..beta..-aminoenone structure.},

  doi          = {10.1021/bi00412a017},

  url          = {https://www.osti.gov/biblio/7163842},
  journal      = {Biochemistry; (United States)},
number       = ,

  volume       = 27:12,

  place        = {United States},

  year         = {Tue Jun 14 00:00:00 EDT 1988},

  month        = {Tue Jun 14 00:00:00 EDT 1988}

}

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Inactivation of the ribonucleoside triphosphate reductase from Lactobacillus leichmannii by 2 prime -chloro-2 prime -deoxyuridine 5 prime -triphosphate: A 3 prime -2 prime hydrogen transfer during the formation of 3 prime -keto-2 prime -deoxyuridine 5 prime -triphosphate

Journal Article Ashley, G; Harris, G; Stubbe, J - Biochemistry; (USA)

The ribonucleoside triphosphate reductase of Lactobacillus leichmannii converts the substrate analogue 2{prime}-chloro-2{prime}-deoxyuridine 5{prime}-triphosphate (C1UTP) into a mixture of 2{prime}-deoxyuridine triphosphate (dUTP) and the unstable product 3{prime}-keto-2{prime}-deoxyuridine triphosphate (3{prime}-keto-dUTP). This ketone can be trapped by reduction with NaBH{sub 4}, producing a 4:1 mixture of xylo-dUTP and dUTP. When (3{prime}-{sup 3}H)C1UTP is treated with enzyme in the presence of NaBH{sub 4}, the isomeric deoxyuridines isolated after alkaline phosphatase treatment retained 15% of the {sup 3}H in C1UTP. Degradation of these isomeric nucleosides has established the location of the {sup 3}H in 3{prime}-keto-dUTP as predominantly 2{prime}(S). The xylo-dU had 98.6% of its labelmore »« less
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Mechanistic studies of ribonucleoside triphosphate reductase from Lactobacillus leichmannii

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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. Furthermore »« less
Interaction of (3'-/sup 3/H)ClUTP with ribonucleotide reductase (RTPR): evidence for an enzyme catalyzed net 1,2 H-shift to produce (2'-/sup 3/H)3'-keta dUTP

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Incubation of (3'-/sup 3/H)ClUTP with prereduced RTPR in the presence of NaBH/sub 4/ resulted in trapping of (/sup 3/H)3'-keto dUTP. Degradation of the reduced ketone by a combination of enzymatic and chemical methods indicated that the hydrogen originally present in the 3' position of ClUTP is on occasion transferred to the 2' position of the 3'-keto dUTP. RTPR catalyzes a net 1,2 H shift. In addition to the production of 3'-keto dUTP, production of dUTP also occurs in 1 out of every 1600 turnovers. Incubation of (2'-/sup 3/H)ClUTP with prereduced RTPR gives results consistent with rapid enolization of 3'-keto dUTPmore »« less
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The redox-active thiols of Escherichia coli ribonucleoside diphosphate reductase and of Lactobacillus leichmannii ribonucleoside triphosphate reductase have been located by a procedure involving (1) prereduction of enzyme with dithiothreitol, (2) specific oxidation of the redox-active thiols by treatment with substrate in the absence of exogenous reductant, (3) alkylation of other thiols with iodoacetamide, and (4) reduction of the disulfides with dithiothreitol and alkylation with (1-/sup 14/C)iodoacetamide. The dithiothreitol-reduce E. coli B1 subunit is able to convert 3 equiv of CDP to dCDP and is labeled with 5.4 equiv of /sup 14/C. Sequencing of tryptic peptides shows that 2.8 equiv ofmore »« less
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