6064-83-1Relevant articles and documents
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Chanley et al.
, p. 4347,4348 (1952)
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Mechanism of inhibition of the class C β-lactamase of Enterobacter cloacae P99 by cyclic acyl phosph(on)ates: Rescue by return
Kaur,Lan,Pratt
, p. 10436 - 10443 (2007/10/03)
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 β-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 μM. 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 β-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 β-lactamase in a time-dependent fashion. It was found, however, to act as a fast reversible inhibitor (Ki = 10 μM). A closer examination of the kinetics of inhibition revealed that both on and off rates (9.8 × 103 s-1 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 β-lactams, the P99 β-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.
Kinetic and Equilibrium Studies of the Copper(II) Promoted Hydrolysis of Salicyl Phosphate. A Rate Acceleration of 1E8 for the Hydrolysis of a Phosphate Monoester Dianion
Hay, Robert W.,Basak, Arup K.,Pujari, Mahesh P.,Perotti, Angelo
, p. 2029 - 2034 (2007/10/02)
The dissociation constants of salicyl phosphate (H3L) at 10 deg C and I= 0.1 mol dm-3 have been determined (pKa values 1.84, 3.57, and 6.18).At a 1:1 ligand to metal ratio the interaction of copper(II) with salicyl phosphate can be described by the equilibria given below. The pKa values for the ionisation processes CuHL - + H+ and - 2- + H+ are 5.1 and 5.9 respectively.Kinetic studies establish that in the copper(II) promoted hydrolysis, the complex - is the active species with kCuL- = 6 * 1E-4 s-1 at 25 deg C and I = 0.1 mol dm-3.Copper(II) ions promote the hydrolysis of the dianion of the phosphate monoester by a factor of ca. 1E8.Previous work in this area had indicated only a small rate acceleration (ca. 10 fold) as comparisons were made between the metal-ion promoted reaction and the intramolecular general acid catalysed hydrolysis of the phosphate monoester dianion.
