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

Abstract

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.