Kinetics and mechanism of inactivation of the RTEM-2 beta-lactamase by phenylpropynal. Identification of the characteristic chromophore.

Abstract beta-Lactamases of all three classes, A, B, and C, are inactivated by phenylpropynal and p-nitrophenylpropynal. The inactivation of RTEM-2 beta-lactamase and of Bacillus cereus beta-lactamase I is accelerated in the presence of A type substrates such as dicloxacillin, quinacillin, and cefoxitin, which are thought to expand or loosen the conformation of these enzymes. In the presence and absence of cefoxitin the inactivation of the RTEM-2 beta-lactamase is first and second order, respectively, in phenylpropynal concentration. The additional phenylpropynal molecule in the latter case may serve the same function as cefoxitin, viz. catalyze access to sensitive functional groups. Correlation of the loss of activity of the RTEM-2 enzyme with the extent of modification suggests that the modification of any one of about four kinetically equivalent groups leads to inactivation. Modification of all of the above mentioned enzymes leads to formation of a characteristic chromophore of unusual stability to nucleophiles, which absorbs maximally between 315 and 320 nm. A consideration of the properties of model compounds demonstrated that the protein-bound chromophore is that of a 1-phenyl-3-imino-1-propen-1-ammonium ion (Formula: see text), formed by reaction of phenylpropynal with two enzymic amine groups, and thus cross-linking the enzyme intramolecularly. Phenylpropynal may be a convenient general reagent for rapid and stable intramolecular cross-linking of proteins through lysine.