title: Elementary steps in the acquisition of Mn2+ by the fosfomycin resistance protein (FosA).
abstract: The fosfomycin resistance protein, FosA, catalyzes the Mn(2+)-dependent addition of glutathione to the antibiotic fosfomycin, (1R,2S)-epoxypropylphosphonic acid, rendering the antibiotic inactive. The enzyme is a homodimer of 16 kDa subunits, each of which contains a single mononuclear metal site. Stopped-flow absorbance/fluorescence spectrometry provides evidence suggesting a complex kinetic mechanism for the acquisition of Mn(2+) by apoFosA. The binding of Mn(H(2)O)(6)(2+) to apoFosA alters the UV absorption and intrinsic fluorescence characteristics of the protein sufficiently to provide sensitive spectroscopic probes of metal binding. The acquisition of metal is shown to be a multistep process involving rapid preequilibrium formation of an initial complex with release of approximately two protons (k(obsd) > or = 800 s(-1)). The initial complex either rapidly dissociates or forms an intermediate coordination complex (k > 300 s(-1)) with rapid isomerization (k > or = 20 s(-1)) to a set of tight protein-metal complexes. The observed bimolecular rate constant for formation of the intermediate coordination complex is 3 x 10(5) M(-1) s(-1). The release of Mn(2+) from the protein is slow (k approximately 10(-2) s(-1)). The kinetic results suggest a more complex chelate effect than is typically observed for metal binding to simple multidentate ligands. Although the addition of the substrate, fosfomycin, has no appreciable effect on the association kinetics of enzyme and metal, it significantly decreases the dissociation rate, suggesting that the substrate interacts directly with the metal center.
authors.affiliation: Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.
authors.forename: B A
authors.initials: BA
authors.lastname: Bernat
authors.forename: R N
authors.initials: RN
authors.lastname: Armstrong
mesh_terms: D001426:Bacterial Proteins; D001665:Binding Sites / Q000187:drug effects; D002614:Chelating Agents / Q000378:metabolism; D004352:Drug Resistance, Microbial; D004492:Edetic Acid / Q000378:metabolism; D005578:Fosfomycin / Q000378:metabolism* / Q000494:pharmacology; D005982:Glutathione Transferase / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008345:Manganese / Q000378:metabolism*; D008667:Metalloproteins / Q000378:metabolism*; D011522:Protons; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
chemical_list: D001426:Bacterial Proteins; D002614:Chelating Agents; D046911:Macromolecular Substances; D008667:Metalloproteins; D011522:Protons; D005578:Fosfomycin; D008345:Manganese; D004492:Edetic Acid; D005982:Glutathione Transferase