Active Site Formation in the Last Stages of Folding of Carbonic Anhydrase

摘要

Carbonic anhydrase is a zinc metalloenzyme catalyzing the exchange reaction: CO2+H2O yields HCO3(-) + H(+). The enzyme consists of a single polypeptide chain with no disulfide links that is folded into a globular structure containing a distinct active site crevice. A unique advantage of carbonic anhydrase for investigation of the principles that govern protein folding, over the usual small proteins that have been investigated (e.g., RNase, lysozyme, cytochrome c), is that the high degree of folding cooperativity that makes direct observation of kinetic intermediates exceptionally difficult is not present in this protein. Intermediates in the folding are known. Our objective in this project is to determine changes in structure and bonding that occur in the active site in the last stages of the folding process. For this purpose, we have modified the carbonic anhydrase by substitution of cobalt(II) for zinc(II). The Co(II) enzyme, unlike all other metal-substituted derivatives, has catalytic properties closely similar to the native zinc enzyme. We have studied the folding of the Co(II) enzyme and have made some comparisons between the folding kinetics of the Co(II) and Zn(II) enzymes. Measurements have been made on both the type I and type II carbonic anhydrases.