The use of cefoxitin, a poor substrate of the RTEM β-lactamase, has allowed the kinetic and spectroscopic characterization of a covalent acyl-enzyme intermediate in the enzyme-catalyzed reaction. The rate of reappearance of catalytic activity in an enzyme sample diluted from an incubation with cefoxitin is nearly identical with the observed kcat. Burst kinetics are observed with this substrate, consistent with the rate-limiting deacylation of the cefoxitinoyl-enzyme. That the reaction intermediate involves a covalent link between enzyme and substrate was shown by gel filtration after rapid denaturation of an enzyme-[14C] cefoxitin reaction at the steady state. Fourier transform infrared measurements indicate that the intermediate is an acyl-enzyme involving a hydroxyl group of the β-lactamase. The evident relationship between the acylation-deacylation sequence of the β-lactamases and the acylation reaction suffered by the D-Ala-D-Ala-carboxypeptidases is discussed.
|Original language||English (US)|
|Number of pages||7|
|State||Published - Jun 1 1980|
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