A simple carbonic anhydrase model which achieves catalytic hydrolysis by the formation of an ‘enzyme–substrate’-like complex

Abstract

One of the attractive features of biomimetic catalysts is their amenability, relative to their enzyme counterparts, to the testing of structural and mechanistic hypotheses. Thus, the reproduction of the active site of an enzyme in a model system provides a tool which is free of much of the complexity of the enzyme. Using this approach, we describe a new model of the active site of the hydrolytic enzyme carbonic anhydrase (CA). The model (1) is composed of a Zn²⁺ complex of the tripodal ligand 1,1,1-tris(aminomethyl)ethane. We find that, in analogy to CA, the complex possesses a water molecule whose pK_a is reduced to 8.0 by coordination to the chelated Zn²⁺ ion. We demonstrate that the complex catalyses the hydrolysis of a model ester substrate (p-nitrophenyl acetate, p-NPA) with a second-order rate constant (k₂) of 0.71 M⁻¹ s⁻¹ (55.0 °C, pH 8.20, ionic strength, I = 0.1 M, aqueous solution), and moreover that it does so with Michaelis–Menten kinetic behaviour (K_m = 7.6 mM; 45.0 °C, pH 8.20, I = 0.1 M, 50% v/v CH₃CN–H₂O). The comparison of these data with those for CA suggests that the hydrophobic cavity and Thr199 residue (which lie adjacent to the active-site of the enzyme) contribute only marginally to the pK_a reduction of the Zn²⁺-bound water molecule. Despite the absence of these moieties, the chelated Zn²⁺ ion is still capable of forming an ‘enzyme–substrate’-like complex, but the stability of the complex is approximately one order of magnitude smaller than that of the enzyme.