Catalysis anhydrase

Carbon atom, 4. See also Atomic orbitals Carbon dioxide hydration, 197-199. See also Carbonic anhydrase Carbonic anhydrase, 197-199,200 Carbonium ion transition state, 154, 159 Carboxypeptidase A, 204-205 Catalysis, general acid, 153,164,169 in carboxypeptidase A, 204-205 free energy surfaces for, 160, 161 in lysozyme, 154... 

A method for the assay (enzyme aetivity) of carbonic anhydrase (Chap. 8. Zn(ll)) uses the catalysis at pH 7.0 of the hydrolysis reaction... 

Redox catalysis Zn, Fe, Cu, Mn, Mo, Co, V Se, Cd, Nl Enzymes (see Table 11.4 for more Information) Reactions with oxygen (Fe, Cu) Oxygen evolution (Mn) Nitrogen fixation (Fe, Mo) Inhibition of llpid peroxidation (Se) Carbonic anhydrase (Cd) Reduction of nucleotides (Co) Reactions with H2 (Nl) Bromoperoxidase activity (V)... 

Carbonic anhydrase presents an instructive case where the catalytic efficiency is so great (kcat > 10 s- ) that proton transfer becomes rate-limiting. The rate was found to depend on the concentration of the protonated form of buffers in the solution. Indeed, Silverman and Tu adduced the first convincing evidence for the role of buffer in carbonic anhydrase catalysis through their observation of an imidazole buffer-dependent enhancement in equilibrium exchanges of oxygen isotope between carbon dioxide and water. The effect is strictly on kcat, and is unaffected because the latter is... 

Importantly, carbonic anhydrase II is one of the most efficient biological catalysts known and it catalyzes the hydration of CO2 with a turnover rate of 10 sec at 25 C (Khalifah, 1971 Steiner et al, 1975). With kcaJKm = 1.5 X 10 sec carbonic anhydrase II is one of a handful of enzymes for which catalysis apparently approaches the limit of diffusion control. Since transfer of the product proton away from the enzyme to bulk solvent comprises a kinetic obstacle [an enzyme-bound group with ap/C, of about 7 cannot transfer a proton to bulk solvent at a rate faster than 10 sec (for a review see Eigen and Hammes, 1963)], the observed turnover rate of 10 sec" requires the participation of buffer in the proton transfer. 

The catalysis of CO2 hydration by carbonic anhydrase II occurs via the two chemically independent steps outlined in Scheme 2 a general mechanistic profile is found in Fig. 23. The first step involves the association of substrate with enzyme and the chemical conversion of substrate into product. The second step is product dissociation and the regeneration of the catalytically active nucleophile zinc hydroxide (Coleman, 1967). Below, we address the structural aspects of zinc coordination in each of these steps. 

There may be two proton transfers in the carbonic anhydrase II-catalyzed mechanism of CO2 hydration that are important in catalysis, and both of these transfers are affected by the active-site zinc ion. The first (intramolecular) proton transfer may actually be a tautomerization between the intermediate and product forms of the bicarbonate anion (Fig. 28). This is believed to be a necessary step in the carbonic anhydrase II mechanism, due to a consideration of the reverse reaction. The cou-lombic attraction between bicarbonate and zinc is optimal when both oxygens of the delocalized anion face zinc, that is, when the bicarbonate anion is oriented with syn stereochemistry toward zinc (this is analogous to a syn-oriented carboxylate-zinc interaction see Fig. 28a). This energetically favorable interaction probably dominates the initial recognition of bicarbonate, but the tautomerization of zinc-bound bicarbonate is subsequently required for turnover in the reverse reaction (Fig. 28b). 

The results of site-directed mutagenesis analysis of zinc ligands of higher plant p-carbonic anhydrase and of P. purpureum carbonic anhydrase have confirmed that zinc is essential for catalysis. X-ray fine structure data indicated that a water molecule is hydrogen bonded to the zinc-ligated Asp-151 and Asp-405. The water molecule is not directly coordinated to the zinc atom. A possible catalytic mechanism of C02 hydration cycle (211) has been proposed as given in Scheme 10. 

Review of the role of water in enzyme catalysis, with carbonic anhydrase as the featured example. 

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). 

Another simple addition reaction is the hydration of C02 to form the bicarbonate ion. Without catalysis the reaction may require several seconds,4 5 the apparent first-order rate constant being -0.03 s 1 at 25°C. Cells must often hasten die process. The specific catalyst carbonic anhydrase is widespread in its distribution... 

Despite the fact that carbonic anhydrase was the first zinc metalloenzyme identified1233 and a good deal is known of its structure, there is still controversy about the nature of the various active-site species and the detailed mechanisms of their action. In particular, the identity of the group with a pXa of 7 that is involved in the mechanism, and the stereochemistry around the zinc ion during catalysis, are still in dispute. The various mechanisms proposed assume either ionization of a histidine imidazole group (bound or not to the zinc) and nucleophilic attack on C02 by the coordinated imidazolate anion,1273,1274 or ionization of the Znn-coordinated water and nucleophilic attack on C02 by OH. 1271 Many papers on this problem have appeared recently and the extensive literature is the subject of the several review articles referred to above. 

Carbonic anhydrase is a zinc(II) metalloenzyme which catalyzes the hydration and dehydration of carbon dioxide, C02+H20 H+ + HC03. 25 As a result there has been considerable interest in the metal ion-promoted hydration of carbonyl substrates as potential model systems for the enzyme. For example, Pocker and Meany519 studied the reversible hydration of 2- and 4-pyridinecarbaldehyde by carbonic anhydrase, zinc(II), cobalt(II), H20 and OH. The catalytic efficiency of bovine carbonic anhydrase is ca. 108 times greater than that of water for hydration of both 2- and 4-pyridinecarbaldehydes. Zinc(II) and cobalt(II) are ca. 107 times more effective than water for the hydration of 2-pyridinecarbaldehyde, but are much less effective with 4-pyridinecarbaldehyde. Presumably in the case of 2-pyridinecarbaldehyde complexes of type (166) are formed in solution. Polarization of the carbonyl group by the metal ion assists nucleophilic attack by water or hydroxide ion. Further studies of this reaction have been made,520,521 but the mechanistic details of the catalysis are unclear. Metal-bound nucleophiles (M—OH or M—OH2) could, for example, be involved in the catalysis. 

There is general agreement that carbonic anhydrase activity is linked to the zinc ion and its ligands. At the active site water is bound to the zinc but the state and involvement of the water in the actual catalysis is a matter of controversy. According to a widely accepted opinion the zinc-bound water is ionized and the activity is a function of pH1S6. ... 

General acid-base catalysis is often the controlling factor in many mechanisms and acts via highly efficient and sometimes intricate proton transfers. Whereas log K versus pH profiles for conventional acid-base catalyzed chemical processes pass through a minimum around pH 7.0, this pH value for enzyme reactions is often the maximum. In enzymes, the transition metal ion Zn2+ usually displays the classic role of a Lewis acid, however, metal-free examples such as lysozyme are known too. Good examples of acid-base catalysis are the mechanisms of carbonic anhydrase II and both heme- and vanadium-containing haloperoxidase. 

A structure-function study of a proton pathway in the y-class carbonic anhydrase from Methanosarcina thermophila was conducted in the work of Tripp and Ferry (2000). Four enzyme glutamate residues were characterized by site-directed mutagenesis. It was shown that Glu 84 and an active site residue, Glu 89, are important for CO2 hydration activity, while external loop residues, Glu 88 and Glu 89 are less important. Glu 84 can be substituted for other ionizable residues with similar pKa values and, therefore, participates in the enzyme catalysis not as a chemical reagent but as a proton shuttle. 

Zinc was demonstrated to be an essential trace element for eukaryotes in 1869 it has subsequently been shown to be essential for all life forms. It generally plays either a catalytic or a structnral role in some metalloproteins that interact with DNA, zinc appears to play both roles. It is an essential constitnent of many enzyme systems, as first demonstrated, for carbonic anhydrase, in 1940. Several hundred zinc metalloenzymes and metaUoproteins have now been recognized, in a wide range of organisms. It generally functions at or near the active site, particnlarly in catalysis of... 

This catalyzed reaction is 10 times as fast as the uncatalyzed one. We will consider the mechanism of carbonic anhydrase catalysis in Chapter 9. Enzymes are highly specific both in the reactions that they catalyze and in their choice of reactants, which are called substrates. An enzyme usually catalyzes a single chemical reaction or a set of closely related reactions. Side reactions leading to the wasteful formation of by-products are rare in enzyme-catalyzed reactions, in contrast with uncatalyzed ones. 

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