Carbonic anhydrase, turnover

Different enzymes exhibit different specific activities and turnover numbers. The specific activity is a measure of enzyme purity and is defined as the number of enzyme units per milligram of protein. During the purification of an enzyme, the specific activity increases, and it reaches its maximum when the enzyme is in the pure state. The turnover number of an enzyme is the maximal number of moles of substrate hydrolyzed per mole of enzyme per unit time [63], For example, carbonic anhydrase, found in red blood cells, is a very active enzyme with a turnover number of 36 X 106/min per enzyme molecule. It catalyzes a very important reaction of reversible hydration of dissolved carbon dioxide in blood to form carbonic acid [57, p. 220],... 

Perhaps the only distinct advantage of enzymic catalysts is their (occasionally) very high turnover rate in situ. Thus, the molar activity (formerly called the turnover number) of some enzymes approaches 36,000,000/min/molecule (7). This latter number pertains to carbonic anhydrase C, the enzyme that converts C02 to HC03 . However, chemists do not need enzymes to convert COz to HCO3-, as long as we are not considering in vivo reactions. Since many enzymes have molar activities as low as 1150/min/molecule, we need not consider molar activities of 100 to 500 (for nonenzymic catalysts) as a severe handicap. It is evident that enzymes and nonenzymic chiral catalysts, rather than being competitors, complement one another. 

Carbonic anhydrase is a metalloprotein with a co-ordinate bonded zinc atom immobilized at three histidine residues (His 94, His 96 and Hisl 19) close to the active site of the enzyme. The catalytic activity of the different isoenzymes varies but cytosolic CA II is notable for its very high turnover number (Kcat) of approximately 1.5 million reactions per second. 

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. 

Subsequent to CO2 association in the hydrophobic pocket, the chemistry of turnover requires the intimate participation of zinc. The role of zinc is to promote a water molecule as a potent nucleophile, and this is a role which the zinc of carbonic anhydrase II shares with the metal ion of the zinc proteases (discussed in the next section). In fact, the zinc of carbonic anhydrase II promotes the ionization of its bound water so that the active enzyme is in the zinc-hydroxide form (Coleman, 1967 Lindskog and Coleman, 1973 Silverman and Lindskog, 1988). Studies of small-molecule complexes yield effective models of the carbonic anhydrase active site which are catalytically active in zinc-hydroxide forms (Woolley, 1975). In addition to its role in promoting a nucleophilic water molecule, the zinc of carbonic anhydrase II is a classical electrophilic catalyst that is, it stabilizes the developing negative charge of the transition state and product bicarbonate anion. This role does not require the inner-sphere interaction of zinc with the substrate C=0 in a precatalytic complex. 

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). 

Carbonic anhydrase of erythrocytes (Mr 30,000) has one of the highest turnover numbers we know of. It catalyzes the reversible hydration of C02 ... 

Carbon monoxide oxidase 893 Carbonic acid, pkCa value of 99 Carbonic anhydrase 443,676 - 678,710 active site structure 679 mechanism 678 turnover number of 458,678 Carbonium ion. See Carbocation 1,1 -Carbonyl-diimidazole 105s Carbonyl group... 

It was once thought that the rate of equilibrium of the catalytic acid and basic groups on an enzyme with the solvent limited the rates of acid- and base-catalyzed reactions to turnover numbers of 103 s 1 or less. This is because the rate constants for the transfer of a proton from the imidazolium ion to water and from water to imidazole are about 2 X 103 s 1. However, protons are transferred between imidazole or imidazolium ion and buffer species in solution with rate constants that are many times higher than this. For example, the rate constants with ATP, which has a pKa similar to imidazole s, are about I0 J s 1 M-1, and the ATP concentration is about 2 mM in the cell. Similarly, several other metabolites that are present at millimolar concentrations have acidic and basic groups that allow catalytic groups on an enzyme to equilibrate with the solvent at 107 to 108 s-1 or faster. Enzyme turnover numbers are usually considerably lower than this, in the range of 10 to 103 s-1, although carbonic anhydrase and catalase have turnover numbers of 106 and 4 X 107 s 1, respectively. 

The catalytic activity of an enzyme is measured by its turnover number, which is defined as the number of substrate molecules acted on by one molecule of enzyme per second. As indicated in Table 24.2, enzymes vary greatly in their turnover number. Most enzymes have values in the 1-20,000 range, but carbonic anhydrase, which catalyzes the reaction of C02 with water to yield bicarbonate ion, acts on 600,000 substrate molecules per second. 

Carbonic anhydrase II, present in human red blood cells (RBCs), catalyzes the reversible hydration of C02. It is one of the most efficient enzymes and only diffusion-limited in its turnover numbers. The catalytic Zn11 is ligated by three histidine residues and OH this ZnOH+ structure renders the zinc center an efficient nucleophile which is able to attack the C02 molecule and capture it in an adjacent hydrophobic pocket. The catalytic mechanism is shown in Figure 9.5. 

A simple calculation reveals that the picture cannot be quite as simple. Carbonic anhydrase has an exceptionally high overall rate of reaction, its turnover number kcat is -5 x 105 s-1 consequently, the rate constants of individual steps must be greater than this number. The acid dissociation of a Zn11 aqua species seems to be inconsistent with this requirement. The dissociation constant fQ can be written as the ratio of forward k and backward kh rate constants [Eq. (9.20)]. 

The uncatalysed reaction is slow(k= 9.5 x 10-2 Lmol 1s 1 at 25°C), however, in the presence of carbonic anhydrase the rate increases to 5 x 107 Lmol 1s 1 which represents 500,000 turnovers per second for each enzyme molecule. Carbonic anhydrase has a globular structure and has a mass of about 29 kDa. The single zinc ion is bound to three nitrogens (from histidine residues) and a water molecule or, as in Fig. 4.18, nearby amino acid occupies the fourth site. 

For example, a lO M solution of carbonic anhydrase catalyzes the formation of 0.6 M H2CO3 per second when it is fully saturated with substrate. Hence, 2 is 6 x lO s b This turnover number is one of the largest known. Each catalyzed reaction takes place in a time equal to 1/ 2 which is 1.7 is for carbonic anhydrase. The turnover numbers of most enzymes with their physiological substrates fall in the range from 1 to 10 per second (Table 8.6). 

Is faster better Restriction endonucleases are, in general, quite slow enzymes with typical turnover numbers of 1 s" f Suppose that endonucleases were faster with turnover numbers similar to those for carbonic anhydrase (10 s i). Would this increased rate be beneficial to host cells, assuming that the fast enzymes have similar levels of specificity ... 

An enzyme electrode is basically a dense package of dialyzer, enzyme reactor, and electrode (detector). Enzymes introduce analytical selectivity due to the specificity of the signal-producing interaction of the enzyme with the analyte. They enhance the equihbrium formation of chemical reactions. For example, splitting of H2O2 is accelerated by a factor of 3 x 10 in the presence of catalase. Turnover numbers can be as fast as 6 X 10 s (carbonic anhydrase) where cat/ m approaches the diffusion limited value of 10 s ... 

Catalysis of carbon dioxide hydration by carbonic anhydrase is the most rapid enz5fmatic reaction known (Table 9.7). It has been shown (Khalifah, 1971) that the Km for the human forms of the enzyme are between 4 and 9 mM, and the catal5dic turnover number, Vmax/ is 8 X 10 (Table 9.7). 

The Idnetic rate constants for CO2 hydration determined in the laboratory in sterile seawater (Table 4.6) are known sufficiently well that this value should create little uncertainty in the above calculation. However, in natural waters the reaction rates may be enzymatically catalyzed. Carbon dioxide hydration catalysis by carbonic anhydrase (CA) is the most powerful enzyme reaction known (see the discussion in Section 9.3). The catal5dic turnover number (the number of moles of substrate reacted, divided by the number of moles of enz5mie present) is 8 x 10 min for CA (Table 9.7), and marine diatoms are loiown to produce carbonic anhydrase (Morel et al, 1994). The calculations presented in Fig. 10.14 indicate that increasing the CO2 hydration rate constant by 10-fold should increase the gas exchange rate of CO2 in the ocean by 10%-50%. 

The fin ax of an enzyme is a measure of how fast the reaction it catalyzes can proceed once the enzyme-substrate complex is formed. This is related to the turnover numberthe number of substrate molecules converted into product per active site at very high substrate concentration. Turnover numbers vary from very high (e.g., 600,000 sec-1 for carbonic anhydrase) to relatively slow (e.g., 0.5 sec-1 for lysozyme). Another name for turnover number is cat, which is related to Em ax and the total enzyme concentration, [E] (measured as number of active sites per unit volume of solution), as follows Em ax = Acat x [E],... 

This is an important process in the transport of CO2 from the tissues to the lungs. If 10.0 p,g of pure carbonic anhydrase catalyzes the hydration of 0.30 g of CO2 in 1 min at 37 °C at Vmax, what is the turnover number (fccat) of carbonic anhydrase (in units of min ) ... 

Enzymes demonstrate both high specificities and significant reaction rate accelerations. The relative values of enzymic over non-enzymic reactions may be from 10 ° to 10 (orotidine decarboxylase) and the turnover numbers range from one catalytic event per minute to 10 per second (hydration of CO2 to HC03 by carbonic anhydrase). The molecular entities of enzymes cover proteins, ribozymes and catalytic antibodies. 

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