Evidence for an initial fast nucleation process in the folding of human carbonic anhydrase I.

Kinetic studies of the folding of carbonic anhydrase have indicated the occurrence of various conformational intermediates. Human carbonic anhydrase I contains a single cysteine residue, Cys-212, which in the native state is unavailable for alkylation. In the unfolded state, it can be specifically modified with iodoacetate. In this study the accessibility of Cys-212 in human carbonic anhydrase I to iodo[2-14C]acetate during the refolding process has been investigated. It is shown that Cys-212 is hidden to the alkylating agent as soon as the refolding is initiated. Since Cys-212 is located in the extensive beta-structure passing through the enzyme, it appears that the Cys-containing beta-strand is part of a rapidly formed nucleation center created during the folding process. This beta-strand (No. 7) together with its neighboring beta-strand (No. 6) constitute the most hydrophobic regions of the enzyme. Because hydrophobic contacts are considered to be important in predicting nucleation sites, these beta-strands probably partake in the formation of the nucleation center. These beta-strands are also partly involved in the bottom region of the active site cavity, indicating that this region is formed during the initial folding events. As a result of this study it was also observed that 2-mercaptoethanol is a potent inhibitor of the enzyme with a K1 = 26 microM at pH 8.0.