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 expressio n 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 solubl e 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 f or full activity, and the Zn(II) ions can be removed by EDTA hom the e nzyme. 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, 6 15, 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 mechan istic studies of the enzyme. Although steady-state and transient-state kinetic studies of the soluble Zn(II) enzyme with nitrocefin as subst rate found no ionizable groups with pK(a) 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 studi es 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.