No abstract

No abstract

No abstract

Hydrogen peroxide, which is a substrate of vanadium bromoperoxidase (V-BrPO), has been shown to be a noncompetitive inhibitor of V-BrPO. Hydrogen peroxide inhibition increases with increasing pH. The inhibition is reversible under the conditions of the initial steady-state kinetic experiments. Analysis of the inhibition constants (KiiH2O2, KisH2O2) versus H+ concentration indicates that an ionizable group with a pKa between 6.5 and 7 is involved in the inhibition. The origin of the oxygen atoms in the dioxygen produced by the V-BrPO-catalyzed bromide-assisted disproportionation of hydrogen peroxide has been shown through H2(18)O2 labeling experiments to originate from the same molecule of hydrogen peroxide. V-BrPO-catalyzed bromination is shown to be an electrophilic (Br+) as opposed to a radical (Br.) process. The stoichiometry of H2O2 consumed to MCD reacted or to O2 produced is reported. The concentration of hydrogen peroxide also affects the competition of dioxygen formation during MCD bromination; competitive dioxygen formation is strongly enhanced at high pH. Turnover of V-BrPO under conditions of very high hydrogen peroxide concentration leads to irreversible inactivation at pH 4 and pH 5. Much less inactivation occurs during turnover at long reaction times at higher pH (> pH 6), and the inactivation can be fully reversed by subsequent addition of vanadate.

A mutation was constructed in the CAP homology domain of yeast topoisomerase II that resulted in hypersensitivity to the intercalating agent N-[4-(9-acridinylamino)-3-methoxy-phenyl]methanesulfonamide and the fluoroquinolone 6,8-difluoro-7-(4-hydroxyphenyl)-1-cyclopropyl-4-quinolone-3-carboxylic acid, but not to etoposide. This mutation, which changes threonine at position 744 to proline, also confers hypersensitivity to anti-bacterial fluoroquinolones. The purified T744P mutant protein had wild type enzymatic activity in the absence of drugs, and no alteration in drug-independent DNA cleavage. Enhanced DNA cleavage in the presence of N-[4-(9-acridinylamino)-3-methoxy-phenyl]methanesulfonamide and fluoroquinolones was observed, in agreement with the results observed in vivo. DNA cleavage was also seen in the presence of norfloxacin and oxolinic acid, two quinolones that are inactive against eukaryotic topoisomerase II. The hypersensitivity was not associated with heat-stable covalent complexes, as was seen in another drug-hypersensitive mutant. Molecular modeling suggests that the mutation in the CAP homology domain may displace amino acids that play important roles in catalysis by topoisomerase II and may explain the drug-hypersensitive phenotype.DNA topoisomerases are enzymes that catalyze changes in the topology of DNA by transiently breaking phosphodiester bonds in the DNA backbone (1). Two major classes of topoisomerases have been identified, type I enzymes, which introduce single strand breaks in DNA, and type II enzymes, which introduce transient double strand breaks. The ability of these enzymes to resolve the winding problems that arise during nucleic acid metabolism render them essential for many of DNA metabolic processes including transcription, DNA replication, and chromosome segregation. Both type I and type II enzymes are ubiquitous among both prokaryotes and eukaryotes, and recent work has led to the discovery of several novel topoisomerases with novel biochemical properties and important specialized physiological roles (reviewed in Ref. 2).In addition to their numerous biological functions, topoisomerases are important targets for both anti-bacterial agents and anti-cancer chemotherapeutic agents (reviewed in Refs. 3-5). Most of the topoisomerase targeting drugs in clinical use are topoisomerase poisons, agents that lead to enhanced levels of an intermediate of the topoisomerase reaction where the enzyme is covalently bound to DNA by a phosphotyrosine linkage and the DNA strand scission has occurred (4). Extensive evidence supports the hypothesis that topoisomerase poisons kill cells because of the DNA damage generated by the covalent complex rather than by depriving cells of an essential enzymatic activity (3, 6). Therefore, resistance to topoisomerase poisons can occur by a change in the level of protein-DNA covalent complexes that arise in the presence of drug, either by reduced activity of the enzyme or by an intrinsic change in sensitivity of the enzyme to the poison.Eukaryotic topoisomeras...