One-step construction of caged carbonic anhydrase I using a ligand-directed acyl imidazole-based protein labeling method

摘要

Caged enzymes whose activities can be controlled by light represent a powerful tool for various biological analyses. However, limited methods are available for the construction of caged proteins and enzymes. We recently developed a novel protein labeling method termed Iigand-directed acyl imidazole (LDAI) chemistry, which allows us to selectively modify natural dihydrofolate reductase and folate receptor in a test tube and in live cell contexts. In this work, we have examined in detail the reaction characteristics of the LDAI chemistry using carbonic anhydrase I (CAI) as a model enzyme. In addition to modifying Lys residues with a carbamate bond, the LDAI method modified Ser and Tyr residues with a carbonate bond. Owing to the relatively labile carbonate bond formed, the LDAI chemistry was demonstrated to be applicable for a rational one-step construction of caged enzymes. This method is simple and based on the transient tethering of an inhibitor with moderate activity that is directed to the active site on an enzyme surface. We successfully showed that the activity of the caged CAI was almost completely suppressed by LDAI-based labeling and fully recovered by photoirradiation in the crude conditions (such as cell lysates) as well as in test tube settings.