Clavulanate inactivation of Staphylococcus aureus beta-lactamase.
The interaction of clavulanic acid with beta-lactamase from Staphylococcus aureus was investigated, particularly with a view to determining whether conformational effects are involved. The inactivation at neutral pH is essentially stoichiometric, leading to an inactive species with an enamine chromophore. Two forms of the enamine were observed, the first-formed having a positive ellipticity with a maximum near 290 nm. This species slowly converted into the stable form of the inactivated enzyme that had a negative ellipticity with a minimum at 275 nm. This change in sign of the ellipticity of the enamine is consistent with the previously proposed cis-trans isomerization of the enamine [Cartwright & Coulson (1979) Nature (London) 278, 360-361). Both the far-u.v.c.d. and the intrinsic viscosity of the inactivated enzyme indicated that negligible change in conformation of the enzyme accompanied inactivation. The rates of inactivation and enamine formation were compared at low temperatures, where the initial rates were slow enough to be monitored. The rate of loss of 95% of the catalytic activity was almost 100-fold faster than the rate of formation of the first-formed enamine species. The remaining 5% activity was lost with a rate comparable with that for formation of the initial enamine. The simplest explanation of these results is that a relatively stable acyl-enzyme intermediate builds up initially and more slowly partitions between turnover (hydrolysis) and enamine formation. The initially formed enamine is in the cis conformation but slowly isomerizes to the more stable trans form.
Rizwi
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Department of Biochemistry, University of Newcastle upon Tyne, U.K.
Tan
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Fink
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Virden
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D000588:Amines / Q000096:biosynthesis; D019818:Clavulanic Acid; D002969:Clavulanic Acids / Q000494:pharmacology*; D003080:Cold Temperature; D006868:Hydrolysis; D007700:Kinetics; D008968:Molecular Conformation; D013056:Spectrophotometry, Ultraviolet; D013211:Staphylococcus aureus / Q000201:enzymology*; D014783:Viscosity; D065093:beta-Lactamase Inhibitors
D000588:Amines; D002969:Clavulanic Acids; D065093:beta-Lactamase Inhibitors; D019818:Clavulanic Acid