Elementary steps in the acquisition of Mn2+ by the fosfomycin resistance protein (FosA)

The fosfomycin resistance protein, FosA, catalyzes the Mn2+-dependent addit ion of glutathione to the antibiotic fosfomycin, (IR,2S)-epoxypropylphospho nic acid, rendering the antibiotic inactive. The enzyme is a homodimer of 1 6 kDa subunits, each of which contains a single mononuclear metal site. Sto pped-flow absorbance/fluorescence spectrometry provides evidence suggesting a complex kinetic mechanism for the acquisition of Mn2+ by apoFosA. The bi nding of Mn(H2O)(6)(2+) to apoFosA alters the UV absorption and intrinsic f luorescence characteristics of the protein sufficiently to provide sensitiv e spectroscopic probes of metal binding. The acquisition of metal is shown to be a multistep process involving rapid preequilibrium formation of an in itial complex with release of approximately two protons (k(obsd) greater th an or equal to 800 s(-1)). The initial complex either rapidly dissociates o r forms an intermediate coordination complex (k > 300 s(-1)) with rapid iso merization (k greater than or equal to 20 s(-1)) to a set of tight protein- metal complexes. The observed bimolecular rate constant for formation of th e intermediate coordination complex is 3 x 10(5) M-1 s(-1). The release of Mn2+ from the protein is slow (k approximate to 10(-2) s(-1)). The kinetic results suggest a more complex chelate effect than is typically observed fo r metal binding to simple multidentate ligands. Although the addition of th e substrate, fosfomycin, has no appreciable effect on the association kinet ics of enzyme and metal, it significantly decreases the dissociation rate, suggesting that the substrate interacts directly with the metal center.