Efficient catalysis by beta-lactamase from Staphylococcus aureus PC1 accompanied by accumulation of an acyl-enzyme.
The pH- and temperature-dependence of steady-state kinetic parameters for 6-beta-(2-furyl)-acryloylamido-penicillanic acid showed it to be a good substrate of staphylococcal PC1 beta-lactamase, and the viscosity-dependence of K(m)/k(cat) indicated that steps up to the formation of the acyl-enzyme were partially diffusion-limited. In the pH range 4-9, a pre-steady-state transient blue shift in the UV absorption spectrum of the bound furyl-acryloylamido chromophore was of constant amplitude and decayed to the spectrum of the product with a first-order rate constant equal to k(cat). The spectrum of the isolated denatured acyl-enzyme was similar to that of the methyl ester of furyl-acryloylpenicilloic acid, pointing to non-covalent interactions with the folded protein, possibly associated with the charge on Glu-166, as the source of the blue-shifted spectrum. Taken together, these results point to a rapid acylation and slower deacylation at Ser-70 and imply that ionization of groups affecting enzyme activity at alkaline pH, for which likely candidates are Lys-73 and Lys-234, affect the rate of deacylation.
Qi
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Department of Biochemistry and Genetics, Medical School, University of Newcastle upon Tyne, U.K.
Virden
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D000215:Acylation; D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D015394:Molecular Structure; D010406:Penicillins; D012997:Solvents; D013056:Spectrophotometry, Ultraviolet; D013211:Staphylococcus aureus / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature; D014783:Viscosity; D001618:beta-Lactamases / Q000737:chemistry / Q000378:metabolism*
D010406:Penicillins; D012997:Solvents; D001618:beta-Lactamases