β-Lactamase Proceeds via an Acyl-Enzyme Intermediate. Interaction of the Escherichia Coli Rtem Enzyme with Cefoxitin

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 k_cat. 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.