Conformational adaptation of RTEM beta-lactamase to cefoxitin

Abstract

Cefoxitin, a poor substrate of the RTEM beta-lactamase (penicillin amido-beta-lactam hydrolase, EC 3.5.2.6), induces a reversible change in the conformation of the enzyme. The change is manifested in gradual loss of catalytic activity and increased susceptibility to proteolytic inactivation. It is prevented by antibodies, which stabilize the native conformation. By contrast, divalent cations, which have no effect on the native enzyme, delay recovery from the cefoxitin-induced state, presumably by reacting with sites made accessible in the partly unfolded enzyme. Prolonged exposure to excess of cefoxitin causes a similar delay. The kinetic evidence, namely, the initial burst of consumption of cefoxitin and the subsequent gradual recovery of activity with better substrates, appears to be consistent with acylation of the active site by cefoxitin followed by a slower deacylation step [Fisher et al. (1980) Biochemistry 19, 2895-2901]. However, additional evidence leads us to conclude that the kinetics observed reflect deformation of the active site, rather than its blockage, by cefoxitin. Of most significance is the transient change in specificity, i. e. a preferential interaction of the recovering enzyme with substrates which are closest in structure to cefoxitin.