Measurements where known and are comparable to those

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measurements, where known, and are comparable to those obtained on the na- tive enzyme. Although affinity constant values reported in the literature were measured under different experimental conditions of, e.g., pH, buffer type, and buffer concentration, several pH-dependent trends are apparent. According to such de- pendences, three classes of inhibitors can be identified 48 (Figure 2.15). In the
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10 4 10 3 10 ' 5 6 7 pH 8 9 67 Figure 2.16 pH dependence of the apparent affinity constants of nitrate for human I (_), bovine II (.6.), and human II (e) carbonic anhydrases. The curves are best-fit curves obtained assuming non-zero affinity of the anion for species I and 3 of Figure 2.9. The best-fit parameters are reported in Table 2.6. Points in parentheses for HCA I reflect possible binding of a second nitrate ion and have been excluded from the fit. 57 first class, the affinity constant, expressed as log K, decreases linearly with increasing pH. Anions that are weak Lewis bases (Cl ~ ,N 3 ,CH 3 COO - , N0 3 - , etc.) behave in this manner, as do neutral ligands like CH 3 0H and aniline. An example is shown in Figure 2.16. A qualitative fit to such curves can be ob- tained using a single pK a . This behavior could be accounted for by assuming that the ligand binds only the low-pH form of the enzyme, in a simplified scheme in which only one pK a value determines the species distribution in CA. We know, however, that the picture is more complex. If the species distribution calculated according to the scheme of Figure 2.9 is assumed to hold, and if it is assumed that only the two water-containing species (1) and (3) can be bound by the ligand, then actual affinity constants can be evaluated for both species (1) and (3)57 (see Table 2.7). Such constants are similar for the three isoen- zymes, whereas the apparent affinity constants at pH 7, for example, mainly depend on the pK a ' s of the coordinated water according to the values of Table 2.5. Therefore, the low-activity species CA I has larger affinity for anions like nitrate (and bicarbonate) than do the high-activity forms at pH 7.
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68 2 I THE REACTION PATHWAYS OF ZINC ENZYMES AND RELATED BIOLOGICAL CATALYSTS Table 2.7 Affinity constants of nitrate for species 1 and.3 a of cobalt(II)-substituted carbonic anhydrases. 57 HCA I 3.74 ± 0.04 2.62 ± 0.06 " As defined in Figure 2.9. BCA II 4.01 ± 0.02 2.56 ± 0.04 HCA II 4.34 ± 0.04 2.61 ± 0.05 A second type of behavior occurs for weak acids like HCN, H 2 S, and aro- matic sulfonamides (ArS0 2 NH 2 ).76,77 Assuming that the anions (conjugated bases) bind the low-pH species of the enzyme, the bell-shaped plot of log K versus pH (Figure 2.15) can be accounted for. In fact, at low pH, the inhibitors are in the protonated form, which is not suitable for metal binding. At high pH the con- centration of the low-pH species of the enzyme decreases. The maximal appar- ent affinity is experimentally halfway between the pK a of the inhibitor and the "pK a " of the enzyme, treated as if it were only one. The same type of curve is also expected if the high-pH species of the enzyme binds the weak acid. Indeed, kinetic measurements seem to favor this hypothesis for sulfonamides.
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