Resonance is caused by substrate exchange and by a

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resonance is caused by substrate exchange and by a paramagnetic contribution due to bonding. The temperature dependence of the linewidth shows that the latter is determined by the dissociation rate. Such a value is only about 2.5 times larger than the overall CO 2 :;;:::: HC0 3 - exchange-rate constant. Therefore the exchange rate between bound and free HC0 3 - is close to the threshold for the rate-limiting step. Such an exchange rate is related to the higher affinity of the substrate and anions in general for type I isoenzymes than for type II iso- enzymes. This behavior can be accounted for in terms of the pK a of coordinated water (see below). C. What Do We Learn from Cobalt Substitution? 1. Acid-base equilibria It is convenient to discuss the cobalt-substituted carbonic anhydrase en- zyme, since its electronic spectra are markedly pH-dependent and easy to mea- sure (Figures 2.7 and 2.8).56,57 The spectra are well-shaped, and a sharp absorp- tion at 640 nm is present at high pH and absent at low pH. Whereas CoHCA I is almost entirely in the low-pH form at pH 5.7, this is not true for the CoBCA II isoenzyme. The acid-base equilibrium for Co-substituted carbonic anhydrase (deprotonation of the metal-coordinated water) involves three species: N" 2 + N-Co-OH / 2 N (2.10) The first equilibrium has never been directly monitored, and the conditions that determine it are quite vague. However, the five-coordinate species has been proposed in HCA I at low pH values. 48 Figure 2.7 at first seems to show isosbestic points * between 16,000 and 18,000 em - 1, so that a single acidic group could * An isosbestic point is a value of frequency where the two species in an A :::='" B equilibrium have the same absorption. As a consequence, all mixtures of A and B also show the same absorption at that frequency, and all the spectra along, e.g., a pH titration from A to B, plotted one on top of the other, cross at the isosbestic point. The presence of isosbestic points thus indicates the presence of only two species in equilib- rium.
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400 200 300 200 100 55 16 18 20 22 16 18 20 22 Figure 2.7 pH-variation of the electronic spectra of cobalt(II)-substituted BCA II (A), HCA II (B), HCA I (C), and BCA III (D). The pH values, in order of increasing 1'15-6, are (A) 5.8, 6.0, 6.3, 6.7, 7.3,7.7,7.9,8.2,8.8; (B) 6.1, 6.6, 7.1, 7.8, 8.3, 8.6,9.5; (C) 5.3, 6.1, 6.6, 7.0, 7.3, 7.5, 7.9,8.4,8.6,9.1,9.6. 56 0.8 E ,5 . 6 E '5 .6 rna, 0.6 0.4 0.2 Figure 2.8 pH dependence of 1'15.6 for cobalt(II)-substituted BCA III (e), HCA II (0), BCA II (£), and HCA I (_) isoenzymes. The high pH limit value of 1'15-6 is normalized to I for each isoen- zyme. 57
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56 2/ THE REACTION PATHWAYS OF ZINC ENZYMES AND RELATED BIOLOGICAL CATALYSTS account for the experimental data. Therefore, CoHCA I would have a pK a of about 8, CoBCA II and CoHCA II a pK a of about 6. .5, and CoBCA III a pK a around 5.5. The analysis of the dependence of the absorbance on pH, however, clearly shows that two apparent pKa's can be extracted from the electronic spec- tra of at least CoCA I and II (Figure 2.8). These kinds of isoenzymes contain at least another histidine in the cavity, which represents another acidic group, with a pK a of about 6.5 in its free state. The interaction between such an acidic
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