title: Thermodynamics of binding of a sulfonamide inhibitor to metal-mutated carbonic anhydrase as studied by affinity capillary electrophoresis.
abstract: By affinity capillary electrophoresis (ACE), the thermodynamic binding constants of a sulfonamide (SA) inhibitor to bovine carbonic anhydrase II (CA) and metal mutated variants (M-CAs) were evaluated. 1-(4-Aminosulfonylphenylazo)-2-naphthol-6,8-disulfonate was used as the SA in the electrophoretic buffer for ACE. The Scatchard analysis of the dependence of the electrophoretic mobility of native CA on the SA concentration provided the binding constant to be Kb=(2.29±0.05)×10(6) M(-1) (at pH8.4, 25°C). On the other hand, apoCA showed far smaller value [Kb=(3.76±0.14)×10(2) M(-1)], suggesting that the coordination of SA to the Zn(II) center controlled the binding thermodynamics. The ACE of M-CAs showed the same behaviors as native CA but with different Kb values. For example, Co-CA adopting the same tetrahedral coordination geometry as native CA exhibited the largest Kb value [(2.55±0.05)×10(6) M(-1)] among the M-CAs. In contrast, Mn- and Ni-CA, which adopted the octahedral coordination geometry, had Kb values that were about two orders of magnitude lower. Because the hydrophobic cavity of CA around the active center pre-organized the orientation of SA, thereby fixing the ligating NH(-) moiety to the apex of the tetrahedron supported by three basal His3 of CA, metals such as Zn and Co at the center of M-CA gave the most stable CA-SA complex. However, pre-organization was not favored for octahedral geometry. Thus, pre-organization of SA was the key to facilitating the tetrahedral coordination geometry of the Zn(II) active center of CA.
authors.affiliation: Graduate School of Environmental Studies, Tohoku University, 6-6-07 Aramaki-Aoba, Aoba-ku, Sendai 980-8579, Japan.
authors.forename: Yosuke
authors.initials: Y
authors.lastname: Sato
authors.affiliation: Graduate School of Environmental Studies, Tohoku University, 6-6-07 Aramaki-Aoba, Aoba-ku, Sendai 980-8579, Japan.
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Hoshino
authors.affiliation: Graduate School of Environmental Studies, Tohoku University, 6-6-07 Aramaki-Aoba, Aoba-ku, Sendai 980-8579, Japan. Electronic address: iki@m.tohoku.ac.jp.
authors.forename: Nobuhiko
authors.initials: N
authors.lastname: Iki
mesh_terms: D000818:Animals; D024402:Carbonic Anhydrase II / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D002257:Carbonic Anhydrase Inhibitors / Q000138:chemical synthesis / Q000737:chemistry*; D002417:Cattle; D019075:Electrophoresis, Capillary; D019216:Metals, Heavy / Q000737:chemistry*; D008956:Models, Chemical; D009282:Naphthalenesulfonates / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D013449:Sulfonamides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: C000610400:1-(4-aminosulfonylphenylazo)-2-naphthol-6,8-disulfonate; D002257:Carbonic Anhydrase Inhibitors; D019216:Metals, Heavy; D009282:Naphthalenesulfonates; D013449:Sulfonamides; D024402:Carbonic Anhydrase II
keywords: Affinity capillary electrophoresis; Carbonic anhydrase; Metal-substituted enzymes; Sulfonamides; Zn(II)