E what do we learn from manganese and cadmium

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E. What Do We Learn from Manganese and Cadmium Substitution? Several studies have been performed on MnCA. Although CA is not the protein for which Mn(II) has been most extensively used as a paramagnetic probe to map substrates and inhibitors within the metal cavity, by measuring the Tn,} values of protons of the inhibitor N-acetyl-sulfanilamide, and by assuming that dipolar contributions are dominant, researchers have mapped the orientation of the inhibitor inside the active cavity (Figure 2.19).92 This orientation is consis- tent with x-ray data on stronger binding sulfonamides. 64 - 66 MnCA is not com- pletely inactive. 13C NMR studies of the CO 2 ;;:::: HC0 3 - interconversion at pH 8.5 showed that the interconversion rate is about 4 percent that of the native enzyme. 93 The TIl and Ti 1 values of H 13C03 - suggest that bicarbonate might be bidentate in the central step of the catalytic cycle. 93 Data from 1l3Cd studies that have been performed on CdBCA II and CdHCA I are consistent with the general picture presented here. 94 The 113Cd chemical shifts are indeed consistent with a donor set of three nitrogens and two oxygens. The cadmium(II) derivative could thus be five-coordinate with two water mole- Figure 2.19 Schematic drawing of the geometric arrangement of the inhibitor N-acetyl- sulfanilamide in the active cavity of manganese(II)-substituted CA, as revealed by IH NMR spectroscopy.92
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IV. ELUCIDATION OF STRUCTURE-FUNCTION RELATIONSHIPS: CARBONIC ANHYDRASE 73 A J 190 Hz B Figure 2.20 113Cd NMR spectra of Cd-substituted bovine carbonic anhydrase II in the presence of 15N-enriched (A) or 14N-enriched (B) benzenesulfonamide inhibitor. 95 400 0, ppm 300 cules, in agreement with the expectation based on its ionic radius being larger than that of zinc(II). The 113Cd signal of CdBCA II in the presence of benzene- sulfonamide enriched in 15N is split into a doublet because of the nitrogen- cadmium coupling (Figure 2.20).95 This result provides direct evidence for metal- nitrogen bonding in sulfonamides, which has been confirmed by x-ray data. 65 F. Catalytic Mechanism All the above structural and kinetic information obtained under a variety of conditions with different metal ions can be used to propose a catalytic cycle for carbonic anhydrase (Figure 2.21), As shown by studies on the pH-dependent properties of native and metal-substituted CAs, both type-I and type-II proteins have two acidic groups, the zinc-coordinated water and a free histidine. At
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74 ry ry (Ny N N N N H N H N H " / +co 2 " / " I N-Zn 0 -----'>. N-Zn 0 -----'>. N-Zn-O 0 ..,.--- ..,.--- / / ,;-0 / ",y- N N N C A C I cfC +;;r 0 Jr ::w B Jr HNy rj ry (+ I HN o N N II N N C N 0" ,;-0 " " / " " / N-Zn-O N-Zn-O 0 N-Zn C / " / I I E / ~I N H H N H H N 0 Jr 1l " / "- 0 +;;r D H I H N H " / N-Zn-O / " N G H -HC0 3 "'--- ---". N" /OH 2 N-Zn / " N 0 I C /~ HO F 0 Figure 2.21 Proposed catalytic cycle of CA. physiological pH the enzyme is essentially in the Zn-OH form (step A in Figure 2.21). A Zn-OH moiety is a relatively good nucleophile, poised for nucleophilic attack on carbon dioxide.
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