title: Further evidence for independent folding of domains in serine proteases.
abstract: The folding pathway of pancreatic serine proteases was clarified from kinetic studies on the refolding of the glutathione-mixed disulfide derivative of bovine neochymotrypsinogen. Neochymotrypsinogen is prepared from a limited proteolysis of native chymotrypsinogen A by cleavage at Tyr146-Thr147 (Duda and Light (1982) J. Biol. Chem. 257, 9866-9871). The mixed disulfide methodology (Odorzynski and Light (1979) J. Biol. Chem. 254, 4291-4295) was necessary to successfully refold chymotrypsinogen and neochymotrypsinogen. Mixtures of the chromatographically purified amino- and carboxyl-terminal polypeptides of neochymotrypsinogen, as the mixed disulfide derivatives, were refolded at varying molar ratios of the polypeptides. The regeneration of native structure was followed as a function of time from activity measurements and from the regain of the molecular weight of the zymogen. The rate data fit first-order kinetics. The kinetic analysis is compatible with a folding mechanism that supports (a) independent folding of the amino- and carboxyl-terminal domains; (b) identical rates of folding of each domain; and (c) the rate-limiting step is the formation of the interdomain disulfide. The formation of a stable complex of the folded domains was favored by complementary hydrophobic and hydrogen bonding interactions and the formation of the last disulfide bond. The geometric arrangement of the active site residues was regained and the zymogen could be converted to the active enzyme, namely, alpha-chymotrypsin.
authors.affiliation: Department of Chemistry, Purdue University, West Lafayette, Indiana 47907.
authors.forename: A
authors.initials: A
authors.lastname: Light
authors.forename: A M
authors.initials: AM
authors.lastname: al-Obeidi
mesh_terms: D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D002918:Chymotrypsin / Q000737:chemistry; D006868:Hydrolysis; D007700:Kinetics; D011487:Protein Conformation; D012697:Serine Endopeptidases / Q000737:chemistry*
chemical_list: D000596:Amino Acids; D012697:Serine Endopeptidases; D002918:Chymotrypsin