Inactivation of the Enterobacter cloacae P99 beta-lactamase by a fluorescent phosphonate: direct detection of ligand binding at the second 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 cloacae 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 group 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 pKa'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].(ABSTRACT TRUNCATED AT 250 WORDS)
Dryjanski
M
M
Department of Chemistry, Wesleyan University, Middletown, Connecticut 06459.
Pratt
R F
RF
D001665:Binding Sites; D003619:Dansyl Compounds / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D016972:Enterobacter cloacae / Q000201:enzymology*; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D063065:Organophosphonates; D013050:Spectrometry, Fluorescence; D065093:beta-Lactamase Inhibitors
C092733:4-nitrophenyl((dansylamido)methyl)phosphonic acid; D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D008024:Ligands; D063065:Organophosphonates; D065093:beta-Lactamase Inhibitors