Trapping the acyl-enzyme intermediate in beta-lactamase I catalysis.
Cryoenzymology techniques were used to facilitate trapping an acyl-enzyme intermediate in beta-lactamase I catalysis. The enzyme (from Bacillus cereus) was investigated in aqueous methanol cryosolvents over the 25 to -75 degrees C range, and was stable and functional in 70% (v/v) methanol at and below 0 degree C. The value of kcat. decreased linearly with increasing methanol concentration, suggesting that water is a reactant in the rate-determining step. In view of this, the lack of incorporation of methanol into the product means that the water molecule involved in the deacylation is shielded from bulk solvent in the enzyme-substrate complex. From the lack of adverse effects of methanol on the catalytic and structural properties of the enzyme we conclude that 70% methanol is a satisfactory cryosolvent system for beta-lactamase I. The acyl-enzyme intermediate from the reaction with 6-beta-(furylacryloyl)amidopenicillanic acid was accumulated in steady-state experiments at -40 degrees C and the reaction was quenched by lowering the pH to 2. H.p.l.c. experiments showed covalent attachment of the penicillin to the enzyme. Digestion by pepsin and trypsin yielded a single labelled peptide fragment; analysis of this peptide was consistent with Ser-70 as the site of attachment.
Cartwright
S J
SJ
Department of Chemistry, University of California, Santa Cruz 95064.
Tan
A K
AK
Fink
A L
AL
D000215:Acylation; D001409:Bacillus cereus / Q000201:enzymology; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003080:Cold Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D000432:Methanol; D012997:Solvents; D013057:Spectrum Analysis; D014867:Water; D001618:beta-Lactamases / Q000378:metabolism*
D012997:Solvents; D014867:Water; D001618:beta-Lactamases; D000432:Methanol