The hydrolysis of an activated ester by a tris(4,5-di-n-propyl-2-imidazolyl)phosphine-Zn2+ complex in neutral micellar medium as a model for carbonic anhydrase

Abstract

The properties of tris(4,5-di-n-propyl-2-imidazolyl)phosphine–M2+ complexes (3–M2+, M = Zn, Co) in neutral micellar media of Brij-35 and Triton X-100 have been studied in water with respect to their quantitative potenti metric titration, Co2+-visible absorption spectra, and ability of the 3–Zn2+ complex to promote the hydrolysis of the activated ester, p-nitrophenyl acetate (PNPA). Potentiometric titration of the 3–M2+(CIO4–)2 complexes in 20 mM Brij-35 media yields a steep titration curve indicative of the cooperative consumption of two hydroxides, with computed pK1 and pK2 values of 8.75 and 6.25, respectively, and the midpoint of the titration curve (pKapp) being 7.50. A similar titration of the Co2+ complex also indicates cooperative consumption of two HO–, and this is tied to the formation of a 4- or 5-coordinate complex, pKapp ~ 7.3–7.4. The cooperativity is explained in terms of sequential replacement of the two CIO4– ions associated with the 3–M2+ to eventually yield 3–M2+–HO–/(HO–(H2O)n) having the first hydroxide ligated to the metal ion and the second associated as an ion pair. The 3–Zn2+ complex catalyzes the hydrolysis of PNPA in 20 mM Brij-35 and 40 mM Triton X-100. Plots of the observed second order rate constant (k2) vs. pH in Brij-35 increase linearly with pH and plateau to a value of k2max = 0.86 M–1 s–1, with a kinetic pKa of 8.7. These data are analyzed by a process wherein the 3–Zn2+–HO– is kinetically active in the rate-limiting step of the reaction, while the ion-paired (HO–(H2O)n) exists as a spectator to the slow step, possibly promoting rapid breakdown of a tetrahedral intermediate. Analysis of the kinetic data in terms of a model that accounts for the partitioning of PNPA between water and hydrophobic micellar pseudophase indicates that the second-order rate constant of the micelle-bound ester is augmented by 45-fold due to loading of the PNPA substrate into the micelle. Key words: Brij-35, TritonX-100, neutral micelle, carbonic anhydrase model, kinetics, potentiometric titrations, catalysis, p-nitrophenyl acetate hydrolysis.