INACTIVATION OF THE ENTEROBACTER-CLOACAE P99 BETA-LACTAMASE BY A FLUORESCENT PHOSPHONATE - DIRECT-DETECTION OF LIGAND-BINDING AT THE 2ND SITE

The synthesis of a fluorescent beta-lactamase inhibitor, p-nitrophenyl [(dansylamido)methyl]phosphonate is described. The compound inactivated the class C beta-lactamase of Enterobacter cloncae P99 with stoichiometric release of p-nitrophenol, presumably, as with other phosphonate inhibitors, by phosphonylation of the active site serine. The inhibited enzyme exhibited typical dansyl fluorescence emission at 533 nm with excitation maxima at 345 and 283 nm; the latter excitation peak probably arises from radiationless energy transfer to the dansyl,coup from aromatic chromophores on the protein-inspection of the crystal structure shows that the closest are tyrosines. The fluorescence of the p-nitrophenyl phosphonate and the inhibited enzyme varied with pH in a very similar fashion, reflecting dissociation of the dimethylammonium ion in the ground state at low pH and of the sulfonamide in the excited state above pH 6. No perturbation of the fluorescence of the inhibited enzyme due to active site functional groups was observed. This may reflect the distance between the dansyl fluorophore and the phosphonyl group and/or the high pK(a)'s of the protonated active site functional groups in the presence of the phosphonate. The addition of certain small molecular weight N-acyl amino acids, of preferred structure D-RCONHCHR'CO2-, to the inhibited enzyme led to an enhancement of dansyl fluorescence intensity and a blue shift in the emission maximum. This suggested that these molecules bind to the beta-lactamase at a site other than the active site and supports previous kinetic data to this effect [Dryjanski, M., & Pratt, R. F., (1995) Biochemistry 34, preceding paper in this issue]. Fluorescence titrations allowed the determination of dissociation constants for these ligands from this second site. The existence of the second site may well relate to the evolutionary relationship of beta-lactamases to bacterial DD-peptidases, the targets of beta-lactam antibiotics.