Cryoenzymology of staphylococcal beta-lactamase: trapping a serine-70-linked acyl-enzyme.
Various cryosolvents were investigated for their suitability in cryoenzymological experiments with beta-lactamase from Staphylococcus aureus PC1. On the basis of the minimal effects on the catalytic and structural properties of the enzyme, ternary solvents containing ethylene glycol, methanol, and water were found most suitable. The interaction of beta-lactamase with a number of substrates was studied at subzero temperatures. In general, the reaction profiles were similar to those in aqueous solution at above-zero temperatures, with the exception of the slower rates. For cephalosporin substrates, such as PADAC, in which the 3'-substituent may leave to form a more stable form of the acyl-enzyme [Faraci, W., & Pratt, R. (1985) Biochemistry 24, 903-910], this intermediate could be readily stabilized at subzero temperatures. At -40 degrees C the slow rate of deacylation in the reaction with the chromophoric substrate 6 beta-[(furylacryloyl)amino]penicillanic acid permitted the acyl-enzyme to be stoichiometrically accumulated. This intermediate was then stabilized at low pH with trifluoroacetic acid. Isolation by centrifugal gel filtration, followed by pepsin digestion, gave a penicilloyl-labeled peptide which was isolated by HPLC. Subsequent trypsinolysis of this peptide gave a single labeled peptide, corresponding to the octapeptide surrounding the active-site serine, Ser-70.
Virden
R
R
Department of Chemistry, University of California, Santa Cruz 95064.
Tan
A K
AK
Fink
A L
AL
D000215:Acylation; D002511:Cephalosporins / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004795:Enzyme Stability; D005615:Freezing; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D010406:Penicillins / Q000378:metabolism; D011487:Protein Conformation; D012694:Serine / Q000378:metabolism; D012997:Solvents; D013210:Staphylococcus / Q000201:enzymology*; D013696:Temperature; D001618:beta-Lactamases / Q000378:metabolism*
D002511:Cephalosporins; D010406:Penicillins; D012997:Solvents; D012694:Serine; D001618:beta-Lactamases