Inactivation of the Enterobacter cloacae P99 .beta.-Lactamase by a Fluorescent Phosphonate: Direct Detection of Ligand Binding at the Second Site

Abstract

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)

Functional evolution of the serine β-lactamase active site

“…To date, however, the position of this binding site on the P99 β-lactamase has not been determined. 45 Analogous secondary binding sites could not be detected on the class A TEM β-lactamase by similar methods. 24 Although such fragments of an ancestral DD-peptidase binding site may still survive in β-lactamases, it is likely that selection of mutants where the extended binding was blocked would also be a significant part of β-lactamase evolution.…”

“…22, 42 Remnants of such extended binding sites may be detectable on present-day β-lactamases. For example, kinetic 43, 44 and physical data, 45 indicate that the P99 β-lactamase has a binding site distinct from the current β-lactamase active site that is specific for structures containing the motif 10 which can be found in the peptidoglycan of gram negative bacteria. Interactions between this site and the β-lactamase site affect substrate turnover at the latter.…”

β-Lactamases: Why and How

“…As far as is known, these specificity elements are also not present in β-lactamases except perhaps in vestigial form. 79,80 The factors described above, taken together, are sufficient to explain why the serine β-lactamases of today have little DDpeptidase activity.…”

Functionalization of α-hydroxyphosphonates as a convenient route toN-tosyl-α-aminophosphonates

“…α-Sulfonamide phosphonates constitute a very interesting class of compounds. They can be potential candidates for fluorescent-β-lactamase 1 and matrix metalloproteinase (MMPs) inhibitors 2 such as compounds containing carboxylate and hydroxamate moieties, which are well known MMP inhibitors. Moreover, these compounds are promising substrates for the synthesis of N -deprotected α-aminophosphonates, which are important isosteres of α-amino acids, possessing a wide range of biological activities.…”