Abstract
We have attempted to elucidate the mechanism of the acquisition of active‐site conformation in enzymes by studying the unfolding and refolding of Co(II) carbonic anhydrase. This enzyme possesses characteristic absorption and CD spectra in the visible region, which reflect primarily the local conformational environment of the active site where the Co(II) is bound. The conformation of the cobalt–protein is found to be almost the same as the native zinc–protein. It has lower thermal stability and unfolds at a lower concentration of guanidinium chloride, even though a single‐phase transition is observed in these denaturations. The unfolding to the random‐coiled protein was followed by various spectroscopic methods which reflect different conformational changes. The results show that separable stages of unfolding are observable for the cobalt–protein. It appears that the collapse of the active‐site conformation, as followed by changes in visible absorption and CD parameters, occurs immediately after the loosening of the molecule. This is followed by the exposure of buried aromatic residues and the unraveling of the backbone secondary structure. However, renaturation of the cobalt–protein results in an inactive protein which has spectroscopic properties significantly different from those of the native cobalt–protein.