Biochemistry: The Chemical Reactions of Living Cells, Volume 2 - D. Metzler 1980

Types of reactions catalyzed by enzymes
Addition and elimination reactions
Carbonic anhydrase

One of the simplest addition reactions is the Hydration of CO₂, leading to The formation of the bicarbonate ion:

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If this reaction is carried out in the absence of a catalyst, it takes several seconds [101]; the apparent first-order rate constant is ~0.03 s^-1 at 25 °C. When this process takes place in Cells, it is frequently accelerated. The specific catalyst, carbonic anhydrase, is a widely distributed enzyme that exhibits particularly high activity in Cells and Tissues involved in Respiration (for example, in erythrocytes). 1 L of mammalian Blood contains 1–2 g of this enzyme, which is a monomeric protein (with a Molecular Weight of ~30,000) containing ~260 Amino Acids and one tightly bound Zn²⁺ ion per molecule. The three-dimensional structures of two major Isoenzymes isolated from human blood have been determined by X-Ray Diffraction [102, 103]. Both molecules have an ellipsoidal shape with dimensions of ~4.1 × 4.1 × 4.7 nm. The zinc atom in each isoenzyme molecule is located in a deep pocket situated ~1.2 nm from the surface and is coordinated by three Histidine side chains and one H₂O molecule or OH^- group. The four ligands form a distorted tetrahedron (Fig. 7-8).

One of the imidazole groups (the His-119 residue), coordinated to the zinc atom, forms a Hydrogen bond with the carboxyl group of the side chain of a Glu residue, and this carboxyl group, in turn, forms an even more extensive network of Hydrogen Bonds, some of which are shown in Fig. 7-8. This structural feature, reminiscent of the charge-transfer system in Chymotrypsin, may enhance the affinity of imidazole-119 for the metal atom and may also influence the catalytic Properties of the enzyme. Other imidazole groups also form hydrogen bonds with groups of the protein molecule.

Carbonic anhydrase is one of the most active Enzymes known. The CO₂ hydration reaction at 25 °C is characterized by a turnover number of ~10⁶ s^-1. The same enzyme catalyzes the hydration of acetaldehyde [Eq. (7-35)], but this reaction proceeds 1,000 times more slowly. The activity is controlled by the ionization state of a group with a pK_a ≈ 7. According to the most widely accepted theory, it is proposed that the zinc ion coordinates a Water molecule and that the Zn—OH₂ complex loses a proton to form Zn⁺—OH (the proton loss process by the Zn—OH₂ complex is characterized by a pK_a ≈ 7, an unusually low pK_a value that may be related to the hydrophobic environment [104, 104a]). Zn⁺—OH is essentially a stabilized hydroxide ion existing at pH values where OH^- is normally present in very low concentrations. It is this hydroxide ion that attacks CO₂ or the aldehyde substrate. Thus, The Role of Zn²⁺ in this enzyme is to generate the attacking base rather than to polarize the carbonyl group. The latter function is performed by the Zn²⁺ ion located in the active center of carboxypeptidase (Chap. 7, Sec. D,4).

FIG. 7-8. The chelated zinc atom in the active center of human carbonic anhydrase B (after Kannan et al. [103]).

Related to the Reactions Catalyzed by carbonic anhydrase is The addition of the amino group of a Hemoglobin molecule to CO₂ to form carbamino groups (—NH—COO^-) (Chap. 4, Sec. D,6).

Box 7-E

Zinc

On average, a human absorbs 10–15 mg of zinc per day³. Despite the fact that zinc is poorly absorbed, its concentration in tissues is relatively high, and this metal is crucially important for The activity of numerous enzymes. The total zinc content in the body of a 70-kg human is 1.4–2.3 g. A typical concentration of Zn²⁺ ions in tissues is 0.3–0.5 mM; an unusually high concentration (~15 mM) is found in the Prostate Gland.

The zinc ion binds much more strongly to most organic ligands than the Mg²⁺ ion does (Table 4-2). It has a filled 3d orbital and tends to form four covalent bonds of tetrahedral Symmetry, frequently with nitrogen- or sulfur-containing ligands. Unlike Mg²⁺, which interacts rapidly and reversibly with enzymes, Zn²⁺ exhibits a strong tendency to form stable bonds within metalloenzymes. The three-dimensional Structure of several metalloenzymes is now known. In all these enzymes, the Zn²⁺ ion in the active center is surrounded by three imidazole groups, while the fourth coordination position remains open for interaction with the substrate. Of considerable interest is also the fact that the second nitrogen atom of the imidazole group in many cases forms a hydrogen bond with the peptide backbone carbonyl group. The same property has been found for the iron atoms of heme Proteins (Fig. 10-1).

Undoubtedly, from a chemical standpoint, Zn²⁺ in enzymes Functions as a Lewis acid, creating a localized center of positive charge near the nucleophilic center of the substrate^в. This function of the metal ion is discussed in Sec. D,4 in connection with carboxypeptidase (Fig. 7-3). Zinc ions are also essential for the function of Thermolysin (Sec. D,4), dipeptidases, alkaline phosphatase (Sec. D,1), RNA polymerases, DNA polymerases^г, carbonic anhydrase (Fig. 7-8), class II aldolases (Sec. K,2,c), certain Alcohol dehydrogenases (Chap. 8, Sec. 3,2), and superoxide dismutase (Box 10-3). Zinc is also known to bind to Insulin hexamers (Fig. 4-13,C).

The unusual protein metallothionein, present in all animal tissues, binds large quantities of various Metal Ions and particularly Zn²⁺. This small protein with a molecular weight of ~6,600 contains 33% Cysteine and binds as many as six metal ions per protein molecule^д. The Physiological Role of metallothionein remains unknown. It may function as a metal ion buffer or serve to scavenge unwanted metals. In its free form, it could potentially act as a redox buffer, similar to Glutathione (Box 7-Ж).

In many carnivorous animals, the reflecting layer behind the retina, the tapetum lucidum, contains crystals of a Zn²⁺-cysteine complex. Because zinc ions are colorless, their presence has frequently gone unnoticed. There is no doubt that zinc ions will yet be discovered in the cells of many other tissues.

Zinc ions in enzymes can frequently be replaced by Mn²⁺, Co²⁺, and other ions without any significant loss of catalytic activity^в,д,е. From the perspective of nutritional issues, it should be noted that Cu²⁺ and the highly toxic Cd²⁺ compete with zinc. The latter accumulates in the renal cortex. Feeding rats and mice a diet containing cadmium at concentrations below those found in human Kidneys leads to a shortened lifespan.

^a O’Dell B. L., Campbell B. J. (1971). Comp. Biochem, 21, 179–216.

^б Liljas A., Rossman M. G. (1974). Annu. Rev. Biochem, 43, 475–507.

^в Mildvan A. S., (1974). Annu. Rev. Biochem., 43, 357–399.

^г Polesz B. J., Seal G., Loeb L. A. (1974). Proc. Nat. Acad. Sci. USA, 71, 4892–4896.

^д Kägi J. H. R., Himmelhoch S. R., Whangler P. D., Bethune J. L., Vallee B. L. (1974). J. Biol. Chem., 249, 3537–3542.

^e Bigbee W. L., Dahlquist F. W. (1974). Biochemistry, 13, 3542–3549.