Carbonic anhydrase turnover rate

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. 

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. 

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. 

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. 

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)]. 

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 ... 

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%. 

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. 

With carbonic anhydrase, oxygen-18 ( 0) in body water also reaches isotopic equilibrium rapidly with the bicarbonate or carbon dioxide (CO2) in the body. The rate of disappearance of deuterium ( H) from the body therefore reflects water turnover, whereas the rate of disappearance of represents water turnover as well as carbon dioxide production (rC02). 

Enzyme activity refers in general to the catalytic ability of an enzyme to increase the rate of a reaction. The amazing rate (36 million molecules per minute) at which one molecule of carbonic anhydrase converts carbon dioxide to carbonic acid was mentioned earlier. This rate, called the turnover number, is one of the highest known for enzyme systems. More common turnover numbers for enzymes are closer to lOVmin, or 1000 reactions per minute. Nevertheless, even such low numbers dramatize the speed with which a small number of enzyme molecules can transform a large number of substrate molecules. i Table 10.3 gives the turnover numbers of several enzymes. 

The overall rate constant for conversion of the E S complex to products E + P is called the turnover number because it represents the number of substrate molecules a single enzyme molecule turns over into product per unit time. A value of about 10 per second is typical although carbonic anhydrase can reach a value of up to 600,000. 

This discrepancy between enzyme turnover and proton exchange presented a puzzle. Enzymes with turnover rates larger than 10 s like carbonic anhydrase and acetylcholinesterase, were dubbed impossible enzymes This is still sometimes reiterated with respect to these and other systems. However most biochemical reactions are studied in the presence of buffers. If the reaction of carbonic anhydrase is studied in the presence of 10 mM buffer with p =8, then the rate constant for proton exchange... 

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