The use of limited proteolysis to probe interdomain and active site regions of beta-galactosidase (Escherichia coli).
Limited proteolysis by pancreatic elastase (EC 3.4.21.36) and chymotrypsin (EC 3.4.21.1) was used to study the domain structure and active site of beta-galactosidase (EC 3.2.1.23) (Escherichia coli). Treatment with elastase resulted in a rapid cleavage between residues Ala-732 and Ala-733. No inactivation accompanied this cleavage suggesting that this bond is in a hinge region of the protein. Some slow cleavages beyond the initial one were observed to occur and were accompanied by inactivation. Treatment of beta-galactosidase with chymotrypsin resulted in cleavages first between Trp-585 and Ser-586 and then between Phe-601 and Cys-602. The first of these cleavages resulted in total inactivation of beta-galactosidase. The presence of monovalent ions or isopropyl-beta-D-thiogalactopyranoside protected against the cleavages but when Mg2+ or Mn2+ was present in the reaction mixture, the bond between Trp-585 and Ser-586 was more susceptible to the action of chymotrypsin. These data demonstrate that the conformation of beta-galactosidase around Trp-585 and Ser-586 is dramatically affected by the binding of ions and isopropyl-beta-D-thiogalactopyranoside. The mutant M15 beta-galactosidase, which is missing residues 11 through 41 and is an inactive dimer rather than an active tetramer, was found to be much more labile to proteases than native beta-galactosidase, but the same initial cleavages were found to occur. In addition, trypsin cleaved the M15 protein between Arg-431 and Trp-432 while native beta-galactosidase was stable to trypsin.
D000906:Antibodies; D001665:Binding Sites; D002918:Chymotrypsin / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D005696:Galactosidases / Q000378:metabolism*; D007544:Isopropyl Thiogalactoside / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D010196:Pancreatic Elastase / Q000378:metabolism; D010446:Peptide Fragments / Q000276:immunology; D010449:Peptide Mapping; D001616:beta-Galactosidase / Q000378:metabolism*
D000906:Antibodies; D010446:Peptide Fragments; D007544:Isopropyl Thiogalactoside; D008345:Manganese; D005696:Galactosidases; D001616:beta-Galactosidase; D002918:Chymotrypsin; D010196:Pancreatic Elastase; D008274:Magnesium