Changes in the coordination geometry of the active-site metal during catalysis of benzylpenicillin hydrolysis by Bacillus cereus beta-lactamase II.
Rapid-scanning stopped-flow spectroscopy (425-700 nm) has been used to study spectral changes in cobalt(II)-substituted Bacillus cereus beta-lactamase II during the binding and hydrolysis of benzylpenicillin. The experiments were carried out in aqueous solution over a temperature range of 3-20 degrees C. Three metallointermediates have been characterized by their visible absorption spectra. Two of them have visible absorption spectra identical with the intermediates ES1 and ES2 previously observed at subzero temperatures in a mixed aqueous/organic solvent [Bicknell, R., & Waley, S.G. (1985) Biochemistry 24, 6876-6887]. In addition, the branched kinetic pathway observed with the zinc(II) and cobalt(II) beta-lactamase II at subzero temperatures has been shown to occur with the cobalt(II)-substituted enzyme in aqueous solution at above-zero temperatures; thus, at pH 6.0 and 3 degrees C, the rate and equilibrium constants are readily determined for the reaction scheme: (Formula: see text). A third transient intermediate (called ES*) was found to precede ES1 in the pre-steady-state time period. The identity of the intermediates formed in aqueous solution with those previously observed in the cryostudy confirms that the mechanism is not changed either by the presence of an organic cosolvent or by subzero temperatures. Further characterization of ES1 and the steady-state intermediate ES2 at subzero temperatures, where their lifetime may be extended for up to several hours, has involved circular and magnetic circular dichroic studies. The magnetic circular dichroic spectra identify changes in the coordination sphere of the active-site metal during catalysis.(ABSTRACT TRUNCATED AT 250 WORDS)
D001409:Bacillus cereus / Q000201:enzymology*; D001665:Binding Sites; D002510:Cephalosporinase / Q000378:metabolism*; D003035:Cobalt; D006868:Hydrolysis; D007700:Kinetics; D008433:Mathematics; D010400:Penicillin G / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D013053:Spectrophotometry; D001618:beta-Lactamases / Q000378:metabolism*
D003035:Cobalt; D002510:Cephalosporinase; D001618:beta-Lactamases; D010400:Penicillin G