Effect of limited proteolysis in the 8th loop of the barrel and of antibodies on porcine pancreas amylase activity.
The porcine pancreatic alpha-amylase is a (beta/alpha)8-barrel protein, containing domains A and B (peptide sequence 1-403) and a distinct C-domain (peptide sequence 404-496). Separation of the terminal C-domain from the A and B domains has been attempted by limited proteolysis in the hinge region. Subtilisin was found to hydrolyse amylase between residues 369 and 370 situated in the loop between the eighth beta-strand and alpha-helix. The cleaved amylase was isolated by chromatofocusing and found to retain about 60% of the activity of the native enzyme, while the isolated fragments were inactive. Antigen binding fragments prepared from polyclonal antibodies to native amylase and the CNBr-fragment P1 (peptide sequence 395-496) respectively, were tested for influence on the enzyme activity. Antibodies directed against P1 had no effect whereas antibodies against the peptide sequence 1-394 and amylase respectively inhibited hydrolysis of substrates having four or more glucose residues but not of shorter oligomaltosides. Crystallographic analysis revealed that changes in the region of residue 369 might affect the conformation of the active site as well as of a second binding site. This site, located on the enzyme surface, is proposed to be required for the hydrolysis of larger substrates.
Desseaux
V
V
BBMN Faculté des Sciences, Université d'Aix-Marseille III, France.
Payan
F
F
Ajandouz
E H
EH
Svensson
B
B
Haser
R
R
Marchis-Mouren
G
G
D000681:Amylases / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D007140:Immunoglobulin Fab Fragments; D007158:Immunologic Techniques; D007525:Isoelectric Focusing; D008958:Models, Molecular; D010179:Pancreas / Q000201:enzymology*; D010446:Peptide Fragments / Q000378:metabolism; D011487:Protein Conformation; D013381:Subtilisins; D013552:Swine; D014961:X-Ray Diffraction; D000520:alpha-Glucosidases / Q000737:chemistry / Q000378:metabolism
D007140:Immunoglobulin Fab Fragments; D010446:Peptide Fragments; D000681:Amylases; D000520:alpha-Glucosidases; D013381:Subtilisins