title: Zinc binding drives the folding and association of the homo-trimeric gamma-carbonic anhydrase from Methanosarcina thermophila.
abstract: Carbonic anhydrase from the archeon Methanosarcina thermophila (Cam) is a homo-trimeric enzyme, the left-handed beta-helical subunits of which bind three catalytic Zn(2+) ions at symmetry-related subunit interfaces. The observation of activity for holo-Cam at nanomolar concentrations provides a minimal estimated free energy of folding and assembly of the trimeric holo-complex of approximately 70 kcal (mol trimer)(-1) at standard state. Although the direct measurement of stability by chemical denaturation was precluded by the irreversible unfolding of the holo-enzyme, the reversible unfolding of metal-free apo-Cam is well described by a three-state model involving the folded apo-trimer, the folded monomer and the unfolded monomer. The monomer is estimated to have a stability of 4.0 +/- 0.3 kcal (mol monomer)(-1). The association to form apo-trimer contributes 13.2 +/- 0.4 kcal (mol trimer)(-1), a value confirmed by analytical ultracentrifugation measurements. Far- and near-UV circular dichroism data show a progressive increase in secondary and tertiary structure as the apo-monomer is converted to holo-trimer. The literature value for the free energy of binding of one Zn(2+) ion to a canonical active site, 16.4 kcal mol(-1), is consistent with the presumption that the >45 kcal (mol trimer)(-1) generated by the binding of three ions represents the major contribution to the stability of the holo-trimeric Cam.
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01605, USA.
authors.forename: B Robert
authors.initials: BR
authors.lastname: Simler
authors.forename: Brandon L
authors.initials: BL
authors.lastname: Doyle
authors.forename: C Robert
authors.initials: CR
authors.lastname: Matthews
mesh_terms: D001665:Binding Sites; D001704:Biopolymers; D002256:Carbonic Anhydrases / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D002942:Circular Dichroism; D004795:Enzyme Stability; D006639:Histidine / Q000737:chemistry; D017020:Methanosarcina / Q000201:enzymology*; D008958:Models, Molecular; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D020033:Protein Isoforms / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics; D014461:Ultracentrifugation; D014508:Urea / Q000494:pharmacology; D015032:Zinc / Q000378:metabolism*
chemical_list: D001704:Biopolymers; D020033:Protein Isoforms; D006639:Histidine; D014508:Urea; D002256:Carbonic Anhydrases; D015032:Zinc