The ph can then be adjusted by appropriate additions

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The pH can then be adjusted by appropriate additions of NaOH. All the mea- surements reported in the literature performed in acetate, phosphate, imidazole, or tris sulfate buffers are affected by the interference of the anion with the metal ion. However, buffer species containing large anions like Hepes (4[(2-hydrox- yethyl)-l-piperazinyl]ethanesulfonic acid) can be used,45 since these anions do not enter the cavity. There are many indications that zinc in the high-pH form of CA is four- coordinate with an OH group in the fourth coordination site. At low pH the enzyme exists in a form that contains coordinated water; the coordination num- ber can be four (one water molecule) or five (two water molecules). Of course, the occurrence of the low-pH species depends on the pKa's of the complex acid- base equilibria. 51
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52 2 I THE REACTION PATHWAYS OF ZINC ENZYMES AND RELATED BIOLOGICAL CATALYSTS B. Steady-State and Equilibrium Kinetics of Carbonic Anhydrase-Catalyzed C0 2 /HCO a - Interconversion The CO 2 ~ HC0 3 - interconversion catalyzed by CA is extremely fast. The usual kinetic parameters describing an enzymatic reaction are the turnover num- ber or kinetic constant for the reaction, k cat , and the Michaelis constant K m . In the simple catalytic scheme ~ k 2 E + S ~ ES --i> E + P, L , where E stands for enzyme, S for substrate, and P for product, K;;; 1 is given by k l /(L 1 + k 2 ). If k 2 is small, k cat = k 2 and K;;; 1 = k]/L I, the latter corre- sponding to the thermodynamic affinity constant of the substrate for the enzyme. The pH dependences 46 of k cat and K m for CO 2 hydration for the high- and low- activity isoenzymes have been determined (Figure 2.2).33,36 It appears that K m is pH-independent, whereas k cat increases with pH, reaching a plateau above pH 8. For bicarbonate dehydration (the reverse of Equation 2.6), H + is a cosub- strate of the enzyme. The pH dependence of kcat/K m for HC0 3 - dehydration is also mainly due to k cat , which shows the same pH profile as that for CO 2 if the experimental kinetic data are divided by the available concentration of the H + cosubstrate. 47 ,4s Further measurements have shown that the pH dependence of k cat reflects at least two ionizations if the measurements are performed in the absence of anions. 49 The value of k cat reaches its maximum at alkaline pH only when buffer concentrations exceed 10 -2 M. 50 In other words, the exchange of the proton with the solvent is the rate-limiting step along the catalytic pathway if relatively high concentrations of proton acceptors and proton donors are not provided by a buffer system. This limit results from the high turnover of the enzyme, which functions at the limit imposed by the diffusion rate of the H + cosubstrate. At high buffer concentration, k cat shows an isotope effect consistent with the occurrence of an internal proton transfer as the new rate-limiting step.
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