Carbonic anhydrase substrate binding

Coleman, J. E. Mechanism of action of carbonic anhydrase, substrate, sulfonamide, and anion binding. J. Biol. Chem. 242, 5212—5219 (1967). 

This ability of carbonic anhydrase to bind larger substrates and catalyze non-physiological reactions makes it likely that a carbonic anhydrase with a new metal ion could also bind substrates and catalyze new chemical reactions. 

Several model systems related to metalloenzymes such as carboxypeptidase and carbonic anhydrase have been reviewed. Breslow contributed a great deal to this field. He showed how to design precise geometries of bis- or trisimidazole derivatives as in natural enzymes. He was able to synthesize a modified cyclodextrin having both a catalytic metal ion moiety and a substrate binding cavity (26). Murakami prepared a novel macrocyclic bisimidazole compound which has also a substrate binding cavity and imidazole ligands for metal ion complexation. Yet the catalytic activities of these model systems are by no means enzymic. 

Fig. 25. (a) Proposed binding of substrate CO2 (the dark stick in the center) in the hydrophobic pocket of carbonic anhydrase II, as calculated in a molecular dynamics simulation. [Reprinted with permission from Liang, J.-V., Lipscomb, W. N. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3675-3679.] (b) Proposed carbonic anhydrase II-CO2 complex, as calculated in an independent investigation (Merz, 1990, 1991, personal communication). 

Examples of other recombinant enzymes in which an alteration using site-directed mutagenesis resulted in altered substrate binding efficiencies, rates of catalysis, or stability include carbonic anhydrase (Alexander, Nair Christianson, 1991), lactate dehydrogenase (Feeney, Clarke Holbrook, 1990), and several industrially important proteases (Wells etal., 1987 Siezenera/., 1991 Teplyakovcra/., 1992 Aehle et al., 1993 Rheinnecker et al., 1994). 

The catalytic mechanism for C02 hydration-dehydration by carbonic anhydrase represents the focal issue of the present discussion. We have to consider two aspects (1) the mode of binding of the C02 substrate at the active site, and (2) the physical-chemical state of ligands on the zinc ion. 

The idea of H2C03 as substrate for carbonic anhydrase is strongly supported by inhibitor binding studies158, 159. Small anion or sulfonamide inhibitors are linked to the zinc ion at high pH. The complex picks up a proton and the inhibitor is bound in a neutral form. 

Enzymes are proteins that act as catalysts for specific biochemical reactions in living systems. The reactants in enz)une-catalyzed reactions are called substrates. Thousands of vital processes in our bodies are catalyzed by many distinct enzymes. For instance, the enzyme carbonic anhydrase catalyzes the combination of CO2 and water (the substrates), facilitating most of the transport of carbon dioxide in the blood. This combination reaction, ordinarily uselessly slow, proceeds rapidly in the presence of carbonic anhydrase a single molecule of this enzyme can promote the conversion of more than 1 million molecules of carbon dioxide each second. Each enzyme is extremely specific, catalyzing only a few closely related reactions—or, in many cases, only one particular reaction—for only certain substrates. Modern theories of enzyme action attribute this to the requirement of very specific matching of shapes (molecular geometries) for a particular substrate to bind to a particular enzyme (Figure 16-19). 

Circular dlchrolsm studies of proteins and nucleic acids are providing useful data on conformations in solution and their modification by various agents. Studies of the binding of N-acetylglucosamine to egg-white lysozyme and of acetazolamide to human carbonic anhydrase exemplify the application of this technique to the measurement of conformational changes induced in enzymes by small molecule substrates and inhibitors. 

Carbonic anhydrase raises the rate of this interconversion 10 million times. Notice that the enzyme itself does not appear as part of the chemical equilibrium, although its name is sometimes written above the arrows. During the reaction, each enzyme molecule combines with its substrates, C02 and H20, and converts them to product, H2C03. The enzyme and product then separate and the enzyme can bind additional substrate molecules and continue to catalyze further conversions. In this way, one molecule of carbonic anhydrase can hydrate 100,000 molecules of C02 per second. 

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