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

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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 .mu.M at pH 8.0.