Such a limit is then 10 4 s 1 whereas the turnover

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Such a limit is then 10 4 s 1, whereas the turnover rate is 10 6 S 1. The presence of buffer can assist in proton transfer at this stage, in such a way that the rate-limiting step becomes the internal proton transfer. The release of H + from the Zn-OH 2 moiety is also the rate-limiting step for the low-activity CA III, as nicely shown by the elec- tronic spectra of CoCA III. These spectra change from the basic form at the beginning of the reaction to the acidic form upon CO 2 addition (Figure 2.23).101 After the interconversion of CO 2 into bicarbonate, there is an accumulation of the CoOH 2 species, the deprotonation of which is slower than the release of HC0 3 -. G. Model Chemistry Some efforts have been reported in the literature to simulate the activity of CA and therefore to obtain further information on the mechanism. The pK a of Zn-OH 2 moieties in various complexes has been studied as discussed in Sec- 0.090 0.070 <J) u C ttl 0.050 .Q (; Ul .Q ttl 0.45 0.40 0.35 0 milliseconds Figure 2.23 Time dependence of E15.6 and E18'1 of cobalt(II)-substituted CA III after addition of CO 2 to a buffered enzyme solution at pH 8. The initial drop of absorbance reflects the accumulation of a CoOH 2 intermediate. 101
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60 'I E () 'I 0 E 40 ME "0 oS 20 665 500 wavelength (nm) 60 400 77 Figure 2.24 Electronic spectra of Co(TPyMA)OH/+ (Table 2.3) at various pH values. to Note the similarity to the electronic spectra of cobalt-substituted carbonic anhydrase at various pH values as re- ported in Figure 2.7. 56 tion IILA. The electronic spectra of some cobalt analogues have been found to be similar. One such example is shown in Figure 2.24; the complex Co(TPyMA)OH 2 2 + (Table 2.3) 10 provides a five-coordinate adduct with a weakly bound axial nitrogen (Figure 2.25A). The interconversion between Co(TPyMA)OH}+ and Co(TPyMA)OH + was studied by electronic spectroscopy (Figure 2.24). Despite the difference in the number of coordinated nitrogens, the difference between the high- and low-pH forms resembles that of the cobalt enzyme (cf. Figure 2.7).10 Table 2.3 shows that only one compound, with zinc(II) as the metal ion, seems to have three nitrogens and a water, whereas all the other models have a higher coordination numberY-17 The simple [Co IlI (NH 3 hOH]2+ complex has been shown to accelerate the formation of bicarbonate (k = 2 x 10 2 MIS 1), but, of course, bicarbonate remains coordinated to the metal because of the kinetic inertness of cobalt(III).102,103 Some relatively ill-defined systems have been reported to have some kind of activity. The ligand shown in Figure 2.25B, with zinc(II) as the metal ion in H 2 0, accelerates the attainment of the equilibrium 104 (2.17)
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78 (A) cyclodextrin (B) (C) Figure 2.25 Some multidentate ligands as models of CA: (A) tris-(3,5-dimethyl-l- pyrazolylmethyl) amine 4 (cobalt[II] complex); (8) tris (4,5- diisopropylimidozal-2yl)phosphine10 4 (zinc[II] complex); (C) bis(histamino) f3-cyclodextrin.
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