Simple carbonic anhydrase model which achieves catalytic hydrolysis by theformation of an 'enzyme-substrate'-like complex

One of the attractive features of biomimetic catalysts is their amenability , relative to their enzyme counterparts, to the testing of structural and m echanistic hypotheses. Thus, the reproduction of the active site of an enzy me in a model system provides a tool which is free of much of the complexit y 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 co mposed of a Zn2+ complex of the tripodal ligand 1,1,1-tris(aminomethyl)etha ne. 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 subs trate (p-nitrophenyl acetate, p-NPA) with a second-order rate constant (k(2 )) of 0.71 M-1 s(-1) (55.0 degreesC, pH 8.20. ionic strength, I = 0.1 M, aq ueous solution), and moreover that it does so with Michaelis-Menten kinetic behaviour (K-m = 7.6 mM; 45.0 degreesC, pH 8.20. I = 0.1 M, 50% v/v CH3CN- H2O). The comparison of these data with those for CA suggests that the hydr ophobic 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 Z n2+ ion is still capable of forming an 'enzyme-substrate'-like complex, but the stability of the complex is approximately one order of magnitude small er than that of the enzyme.