Inactivation of Ribonucleotide Reductase by (E)-2‘-Fluoromethylene-2‘-deoxycytidine 5‘-Diphosphate: A Paradigm for Nucleotide Mechanism-Based Inhibitors
| Autor: | D J Silva, Guixue Yu, W. A. Van Der Donk, R J Resvick, D P Matthews, E Wagner, JoAnne Stubbe, Esa T. Jarvi, James R. McCarthy |
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| Rok vydání: | 1996 |
| Předmět: |
Alkylation
Stereochemistry Molecular Sequence Data Antineoplastic Agents medicine.disease_cause Biochemistry Dithiothreitol Mice chemistry.chemical_compound Isomerism Ribonucleotide Reductases Escherichia coli medicine Animals Nucleotide Enzyme Inhibitors chemistry.chemical_classification Base Sequence Electron Spin Resonance Spectroscopy Pigments Biological Amino acid Kinetics Enzyme Ribonucleotide reductase Models Chemical chemistry Deoxycytosine Nucleotides Mutation Cytosine |
| Zdroj: | Biochemistry. 35:8381-8391 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi960190j |
| Popis: | Ribonucleotide reductase (RDPR) from Escherichia coli catalyzes the conversion of nucleotides to deoxynucleotides and is composed of two homodimeric subunits: R1 and R2. (E)- and (Z)-2'-fluoromethylene-2'-deoxycytidine 5'-diphosphate (FMCDP) are time dependent inactivators of this protein, with approximately 1.5 equiv being sufficient for complete loss of catalytic activity. Inactivation results from loss of the essential tyrosyl radical on R2 and alkylation of R1. Studies using electron spin resonance spectroscopy reveal that tyrosyl radical loss is accompanied by formation of a new, substrate-based radical. Experiments using [6'-14C]-(E)-FMCDP and [5-3H]-(E)-FMCDP reveal that alkylation of R1 is accompanied by release of 0.5 equiv of cytosine and 1.4 equiv of fluoride ion. When R1 is denatured subsequent to inactivation, approximately 1 equiv of label per R1 is observed only in studies carried out with [14C]FMCDP. Under these same conditions with [3H]FMCDP, 1.5 equiv of radiolabel is detected as cytosine. Inactivation of R1 thus results from alkylation by the sugar moiety of FMCDP. While studies to isolate the alkylated amino acid on R1 were unsuccessful, studies using a variety of site-directed mutants of R1 (C462S, C225S, C754/759S, C439S, and E441Q) indicate that E441 or possibly C439 is the modified residue. Inactivation is accompanied by rapid formation of a new chromophore with a lambda max at 334 nm. Dithiothreitol does not protect the enzyme against inactivation by FMCDP, although it does prevent chromophore formation. Two possible mechanisms are proposed to accommodate these experimental observations. |
| Databáze: | OpenAIRE |
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