Title

Zinc binding drives the folding and association of the homo-trimeric gamma-carbonic anhydrase from Methanosarcina thermophila

UMMS Affiliation

Department of Biochemistry and Molecular Pharmacology

Publication Date

3-31-2004

Document Type

Article

Subjects

Binding Sites; Biopolymers; Carbonic Anhydrases; purification; Circular Dichroism; Enzyme Stability; Histidine; Methanosarcina; Models, Molecular; Protein Conformation; Protein Denaturation; *Protein Folding; Protein Isoforms; Protein Structure, Secondary; Protein Structure, Tertiary; Spectrophotometry, Ultraviolet; Thermodynamics; Ultracentrifugation; Urea; Zinc

Disciplines

Life Sciences | Medicine and Health Sciences

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.

Rights and Permissions

Citation: Protein Eng Des Sel. 2004 Mar;17(3):285-91. Epub 2004 Mar 29. Link to article on publisher's site

DOI of Published Version

10.1093/protein/gzh027

Related Resources

Link to Article in PubMed

Journal/Book/Conference Title

Protein engineering, design and selection : PEDS

PubMed ID

15051865