| Part I. The inactivation of (beta)-lactamases by phenylpropynal (PPL) and p-nitrophenylpropynal (NPPL) was investigated, in particular that of E. coli TEM-2 (TEM) by PPL. The irreversible inactivation of TEM by PPL both in the absence and presence of a poor substrate, cefoxitan, involves lysine modification resulting in formation of vinylogous amidines. This type of chromophoric species ((lamda)(,max) (TURN) 316 nm) is obtained when two lysine residues interact with one molecule of PPL resulting in an intramolecularly cross-linked enzyme. Of the six PPL-TEM cyanine-like adducts possible per enzyme molecule only one affects activity. The critical pair in each inactivated enzyme molecule may or may not contain the same two lysine residues.;Enzyme conformation plays a significant role in the inhibition of TEM by PPL. During inactivation of TEM alone, one molecule of PPL reversibly binds (probably as an imine) causing a conformational change which allows a second molecule of inhibitor to bind irreversibly and totally inactivate the enzyme. Incubation of TEM with cefoxitin eliminates the requirement for the reversible PPL-TEM interaction, since upon binding cefoxitin TEM also assumes a PPL-sensitive conformation. The cefoxitin-induced conformation is (TURN) 4 to 5 times more susceptible to inactivation than the PPL-induced species. Inactivation is most likely due to the bridging of a critical pair of lysines which prevents the now conformationally-restricted enzyme from adopting a conformationally active form.;Part II. The kinetics and mechanism of the Bamberger cleavage of imidazole by diethyl pyrocarbonate in dilute aqueous solution have been studied. The cleavage yields have been measured as a function of reagent concentrations and of pH and the cleavage rates have been measured as a function of reagent concentrations. At low diethyl pyrocarbonate concentrations (< 10 mM) in phosphate buffer at pH 6.0 carbethoxylation of N-carbethoxyimidazole is rate-determining to the cleavage while at higher diethyl pyrocarbonate concentrations breakdown of an intermediate becomes rate-determining. The intermediate has been shown to be 2-hydroxyl-1,3-dicarbethoxy-4-imidazoline, an unusually stable (t(, 1/2) (TURNEQ) 2 min at pH 6.0) hydroxyl substituted carboxylic acid derivative tetrahedral adduct. |
