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

Abstract

Carbonic anhydrase from the archeon Methanosarcina thermophila (Cam) is a homo-trimeric enzyme, the left-handed β-helical subunits of which bind three catalytic Zn2+ 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 ∼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 holoenzyme, 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 Zn2+ 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.