The use of limited proteolysis to probe interdomain and active site regions of beta-galactosidase (Escherichia coli).

摘要: Limited proteolysis by pancreatic elastase (EC 3.4.21.36) and chymotrypsin 3.4.21.1) was used to study the domain structure active site of beta-galactosidase 3.2.1.23) (Escherichia coli). Treatment with resulted in a rapid cleavage between residues Ala-732 Ala-733. No inactivation accompanied this suggesting that bond is hinge region protein. Some slow cleavages beyond initial one were observed occur inactivation. first Trp-585 Ser-586 then Phe-601 Cys-602. The these total beta-galactosidase. presence monovalent ions or isopropyl-beta-D-thiogalactopyranoside protected against but when Mg2+ Mn2+ present reaction mixture, more susceptible action chymotrypsin. These data demonstrate conformation around dramatically affected binding isopropyl-beta-D-thiogalactopyranoside. mutant M15 beta-galactosidase, which missing 11 through 41 an inactive dimer rather than tetramer, found be much labile proteases native same occur. In addition, trypsin cleaved protein Arg-431 Trp-432 while stable trypsin.