The role of the metal ion in the refolding of denatured bovine Co(II)-carbonic anhydrase II.
Conditions for reactivation of guanidine-HCl-denatured bovine Co(II)-carbonic anhydrase II are given. The renaturation is accompanied by recovery of the native Co(II)-spectrum of the enzyme. After studying the kinetics of the renaturation process, the metal ion involvement in the refolding pathway can be summarized as follows: (1) Formation of an inactive Co(II)-intermediate with the metal ion firmly bound. No native Co(II)-spectrum is observed in this state, probably due to octahedral coordination of the metal ion in this intermediate. (2) Formation of an inactive Co(II)-intermediate with a native Co(II)-spectrum. The final tetrahedral coordination of the metal ion seems to have been formed in this state. (3) Formation of the active conformation of the enzyme. A functioning active-site is formed after some rearrangements of the polypeptide chain. This isomerisation step does not need to be preceded by formation of the intermediate with a native Co(II)-spectrum. Coordination of Co2+ in a native-like manner is, however, a prerequisite for enzymic activity. It is tentatively suggested that the metal ion is involved in stabilizing a nucleation structure formed at the bottom of the active centre. This probably occurs through binding of Co2+ to some or all of its histidyl ligands in this region after an early structuration of the metal ion binding site. The mechanisms of Co2+ appear to be similar for the refolding enzyme and the native apoenzyme, inferring that the binding site formed as a result of the nucleation process probably has the same structure as in the native conformation.
Bergenhem
N
N
IFM-Department of Chemistry, Linköping University, Sweden.
Carlsson
U
U
D000818:Animals; D001051:Apoenzymes / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D002256:Carbonic Anhydrases / Q000378:metabolism; D002417:Cattle; D003035:Cobalt / Q000378:metabolism / Q000494:pharmacology; D019791:Guanidine; D006146:Guanidines; D007700:Kinetics; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation; D013053:Spectrophotometry; D015032:Zinc / Q000378:metabolism / Q000494:pharmacology
D001051:Apoenzymes; D006146:Guanidines; D003035:Cobalt; D002256:Carbonic Anhydrases; D015032:Zinc; D019791:Guanidine