Mechanism of Inhibition of the Class C β-Lactamase of Enterobacter cloacae P99 by Cyclic Acyl Phosph(on)ates:  Rescue by Return

Abstract

As previously described (Pratt, R. F.; Hammar, N. J. J. Am. Chem. Soc. 1998, 120, 3004.), 1-hydroxy-4,5-benzo-2,6-dioxaphosphorinone(3)-1-oxide (salicyloyl cyclic phosphate) inactivates the class C beta-lactamase of Enterobacter cloacae P99 in a covalent fashion. The inactivated enzyme slowly reverts to the active form. This paper shows that reactivation involves a recyclization reaction that regenerates salicyloyl cyclic phosphate rather than hydrolysis of the covalent intermediate. The inactivation, therefore, is a slowly reversible covalent modification of the active site. The thermodynamic dissociation constant of the inhibitor from the inactivated enzyme is 0.16 microM. Treatment of the inactivated enzyme with alkali does not produce salicylic acid but does, after subsequent acid hydrolysis, yield one molar equivalent of lysinoalanine. This result proves that salicyloyl cyclic phosphate inactivates the enzyme by (slowly reversible) phosphorylation of the active site serine residue. This result contrasts sharply with the behavior of acyclic acyl phosphates which transiently inactivate the P99 beta-lactamase by acylation (Li, N.; Pratt, R. F. J. Am. Chem. Soc. 1998, 120, 4264.). This chemoselectivity difference is explored by means of molecular modeling. Rather counterintuitively, in view of the relative susceptibility of phosphates and phosphonates to nucleophilic attack at phosphorus, 1-hydroxy-4,5-benzo-2-oxaphosphorinanone(3)-1-oxide, the phosphonate analogue of salicyloyl cyclic phosphate, did not appear to inactivate the P99 beta-lactamase in a time-dependent fashion. It was found, however, to act as a fast reversible inhibitor (K(i) = 10 microM). A closer examination of the kinetics of inhibition revealed that both on and off rates (9.8 x 10(3) s(-1) x M(-1) and 0.098 s(-1), respectively) were much slower than expected for noncovalent binding. This result strongly indicates that the inhibition reaction of the phosphonate also involves phosphylation of the active site. Hence, unlike the situation with bacterial DD-peptidases covalently inactivated by beta-lactams, the P99 beta-lactamase inactivated by the above cyclic acyl phosph(on)ates can be rescued by return. Elimination of the recyclization reaction would lead to more effective inhibitors.

Structural Insight into Potent Broad-Spectrum Inhibition with Reversible Recyclization Mechanism: Avibactam in Complex with CTX-M-15 and Pseudomonas aeruginosa AmpC β-Lactamases

“…The angle between the carbamoyl plane and N6 is 113.4°, making it within the Burgi-Dunitz trajectory (44) for a nucleophilic attack. Similar intramolecular geometry was also observed in the crystal structures of 5,5-trans-lactone inhibitors, which show reversible acylation for porcine pancreatic elastase acyl-enzyme (45) as well as predicted reversible acylation by cyclic acyl phosphates (46). The disposition of residues around the N6 nucleophile in the two acylated structures suggests that Ser130 and Tyr150 are likely to participate as general bases for recyclization in class A and class C, respectively ( Fig.…”

Inhibition of β-Lactamases by Monocyclic Acyl Phosph(on)ates

“…This is suggestive of hydrolysis by water/hydroxide attack at the acyl rather than the phosphyl group since attack at the latter should be significantly faster for the phosphonate. The hydrolysis of 4 under these conditions, for example, was some seven times faster than that of 3, and hydrolysis of the former did involve phosphyl transfer (9). Solvolysis of 6 for 30 min at room temperature in aqueous alkaline methanol (80% methanol, 0.1 M NaOH) yielded a product with one methyl group singlet resonance at δ3.35 in the 1 H NMR spectrum.…”

“…Nucleophilic attack at the acyl group would yield a methyl singlet, whereas attack at phosphorus would yield a methyl doublet; a methyl carboxylate would also hydrolyze within the observed time frame under these conditions, whereas a methyl phosphate would not. The conclusion of attack at carbon has also been drawn for 3 (9). The rate constant for hydrolysis of 5 was significantly smaller than for 6 (viz.…”

“…For example, the half-life of the benzoyl derivative of the Enterobacter cloacae P99 enzyme is around 1 min at 25°C. Extension of this lifetime by structural perturbation is likely to be possible.The cyclic phosph(on)ates 3 and 4 have also been studied in this laboratory (8,9). They are notable in being reversible covalent inhibitors of the P99 -lactamase (Scheme 1) but not substrates (i.e., the enzyme does not catalyze their hydrolysis).…”

Modeling kinase–substrate specificity: implication of the distance between substrate nucleophilic oxygen and attacked phosphorus of ATP analog on binding affinity

“…The reaction mechanism of phosphoryl transfer reactions can favor either an associative pathway that involves a pentacoordinate phosphorus intermediate or dissociative pathway with a triganal planar metaphosphate intermediate [10]. The existence of both pentacoordinate phosphorus intermediate and metaphosphate intermediate has been substantiated by experimental or theoretical methods [11][12][13][14][15][16][17]. Our previous studies on N-phosphoryl amino acid have also proved that many biomimetic reactions preceded through a pentacoordinate phosphorus intermediate [18][19][20][21][22].…”