Purification, characterization, and kinetic studies of a soluble Bacteroides fragilis metallo-beta-lactamase that provides multiple antibiotic resistance.
Resistance to multiple beta-lactam antibiotics traced to the expression of Zn(II) requiring metallo-beta-lactamases has emerged in clinical isolates of several bacterial strains including Bacteroides fragilis, a pathogen commonly found in suppurative/surgical infections. A soluble B. fragilis metallo-beta-lactamase has been purified to homogeneity from the cell growth medium after expression as a secretory protein in Escherichia coli. The enzyme requires two tightly bound Zn(II) ions for full activity, and the Zn(II) ions can be removed by EDTA from the enzyme. The apoenzyme is reactivated by stoichiometric amounts of Zn(II) and Co(II) ions. The Co(II)-substituted enzyme exhibits a UV-visible spectrum characterized by strong Co(II) d-d transitions at 510, 548, 615, and 635 nm and an EPR spectrum with g values of 5. 52, 4.25, and 2.01: features that serve as useful spectroscopic handles for the mechanistic studies of the enzyme. Although steady-state and transient-state kinetic studies of the soluble Zn(II) enzyme with nitrocefin as substrate found no ionizable groups with pKa values between 5.25 and 10.0 involved in catalysis, a kinetically significant proton transfer step in turnover was implicated by studies in deuterium oxide. These studies also detected the accumulation of an enzyme-bound intermediate and provide the basis for a minimal kinetic scheme describing metallo-beta-lactamase-catalyzed nitrocefin hydrolysis.
Department of Chemistry, Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Z
Z
Wang
S J
SJ
Benkovic
D001441:Bacteroides fragilis / Q000201:enzymology*; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D002511:Cephalosporins / Q000378:metabolism; D017931:DNA Primers; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008670:Metals / Q000378:metabolism; D012995:Solubility; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D001618:beta-Lactamases / Q000302:isolation & purification / Q000378:metabolism*
D002511:Cephalosporins; D017931:DNA Primers; D008670:Metals; D001618:beta-Lactamases; C021720:nitrocefin