Novel reaction of elastase with cephalosporin β-lactams

Porcine pancreatic elastase (PPE) was inactivated by two cephalosporin β-lactams, 3-acetoxymethyl-7-α-chloro-3-cephem-4-carboxylate-1,1-dioxide t-butylester (I) and its 7-α-methoxy analog (II) with the first-order rate constants for inactivation, 0.023 and 0.018 s-1 respectively at pH 7.4, 250C. The inhibition was caused by stoichiometric binding of the compounds with PPE (KI, 80 and 30 nM at pH 7.4, respectively) followed by acylation of the active site serine with opening of the lactam ring. PPE inactivated by II (E-II) spontaneously regenerated enzyme activity with a t1/2 of 100 min at both pH 7.4 and 5.0. The reactivation of E-II was slowed with 1% SDS. The major 14C-labeled tryptic peptide of PPE modified with [14C]MeO-labeled II had the amino acid composition of the sequence Ser182 to Arg211. PPE inactivation with I did not reactivate but showed a time-dependent resistance to reactivation by treatment with 0.5 M NH2OH at pH 7.5 and 370C for 10 min. The acid hydrolyzate of PPE-I contained 5 residues of histidine/mole rather than 6 for native PPE. PPE crystals soaked with I in 35% PEG 4000, 0.1 M NaOAc, pH 5.0 were subjected to high resolution x-ray diffraction analysis. The cross-linking of the active site at Ser188 OH and His45 N2 by a 2-substituted, 5-methylene-1,3-thiazine dioxide was clearly demonstrated