title: Revisiting zinc coordination in human carbonic anhydrase II.
abstract: Carbonic anhydrase (CA, general abbreviation for human carbonic anhydrase II) is a well-studied, zinc-dependent metalloenzyme that catalyzes hydrolysis of carbon dioxide to the bicarbonate ion. The apo-form of CA (apoCA, CA where Zn(2+) ion has been removed) is relatively easy to generate, and reconstitution of the human erythrocyte CA has been initially investigated. In the past, these studies have continually relied on equilibrium dialysis measurements to ascertain an extremely strong association constant (K(a) ≈ 1.2 × 10(12)) for Zn(2+). However, new reactivity data and isothermal titration calorimetry (ITC) data reported herein call that number into question. As shown in the ITC experiments, the catalytic site binds a stoichiometric quantity of Zn(2+) with a strong equilibrium constant (K(a) ≈ 2 × 10(9)) that is 3 orders of magnitude lower than the previously established value. Thermodynamic parameters associated with Zn(2+) binding to apoCA are unraveled from a series of complex equilibria associated with the in vitro metal binding event. This in-depth analysis adds clarity to the complex ion chemistry associated with zinc binding to carbonic anhydrase and validates thermochemical methods that accurately measure association constants and thermodynamic parameters for complex-ion and coordination chemistry observed in vitro. Additionally, the zinc sites in both the as-isolated and the reconstituted ZnCA (active CA containing a mononuclear Zn(2+) center) were probed using X-ray absorption spectroscopy. Both X-ray absorption near edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) analyses indicate the zinc center in the reconstituted carbonic anhydrase is nearly identical to that of the as-isolated protein and confirm the notion that the metal binding data reported herein is the reconstitution of the zinc active site of human CA II.
authors.affiliation: Department of Chemistry, Mississippi State University, Mississippi State, Mississippi 39762, USA.
authors.forename: He
authors.initials: H
authors.lastname: Song
authors.forename: David L
authors.initials: DL
authors.lastname: Wilson
authors.forename: Erik R
authors.initials: ER
authors.lastname: Farquhar
authors.forename: Edwin A
authors.initials: EA
authors.lastname: Lewis
authors.forename: Joseph P
authors.initials: JP
authors.lastname: Emerson
mesh_terms: D001051:Apoenzymes / Q000737:chemistry / Q000378:metabolism; D024402:Carbonic Anhydrase II / Q000737:chemistry* / Q000378:metabolism*; D020134:Catalytic Domain; D006801:Humans; D008958:Models, Molecular; D013696:Temperature; D013816:Thermodynamics; D015032:Zinc / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D001051:Apoenzymes; D024402:Carbonic Anhydrase II; D015032:Zinc