title: High kinetic stability of Zn(II) coordinated by the tris(histidine) unit of carbonic anhydrase towards solvolytic dissociation studied by affinity capillary electrophoresis.
abstract: Solvolytic dissociation rate constants (kd) of bovine carbonic anhydrase II (CA) and its metallovariants (M-CAs, M=Co(II), Ni(II), Cu(II), Zn(II), and Cd(II)) were estimated by a ligand substitution reaction, which was monitored by affinity capillary electrophoresis to selectively detect the undissociated CAs in the reaction mixture. Using EDTA as the competing ligand for Zn-CA, the dissociation followed the unimolecular nucleophilic substitution (SN1) mechanism with kd=1.0×10(-7)s(-1) (pH7.4, 25°C). The corresponding solvolysis half-life (t1/2) was 80days, showing the exceptionally high kinetic stability of t Zn-CA, in contrast to the highly labile [Zn(II)(H2O)6](2+), where the water exchange rate (kex) is high. This behavior is attributed to the tetrahedral coordination geometry supported by the tris(histidine) unit (His3) of CA. In the case of Co-CA, it showed a somewhat larger kd value (5.7×10(-7)s(-1), pH7.4, 25°C) even though it shares the same tetrahedral coordination environment with Zn-CA, suggesting that the d(7) electronic configuration of Co(II) in the transition state of the dissociation is stabilized by the ligand field. Among M-CAs, only Ni-CA showed a bimolecular nucleophilic substitution (SN2) reaction path in its reaction with EDTA, implying that the large coordination number (6) of Ni(II) in Ni-CA allows EDTA to form an EDTA-Ni-CA intermediate. Overall, kd values roughly correlated with kex values among M-CAs, with the kd value of Zn-CA deviating strongly from the trend and highlighting the exceptionally high kinetic stabilization of Zn-CA by the His3 unit.
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@tohoku.ac.jp.
authors.forename: Nobuhiko
authors.initials: N
authors.lastname: Iki
mesh_terms: D000818:Animals; D002256:Carbonic Anhydrases / Q000737:chemistry* / Q000378:metabolism; D002417:Cattle; D019075:Electrophoresis, Capillary; D004795:Enzyme Stability; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D015032:Zinc / Q000737:chemistry* / Q000378:metabolism
chemical_list: D006639:Histidine; D002256:Carbonic Anhydrases; D015032:Zinc
keywords: Affinity capillary electrophoresis; Carbonic anhydrase; Metal-substituted enzymes; Sulfonamides; Zn(II)