Spectrofluorimetric investigation of the interactions between the subunits of bovine pancreatic procarboxypeptidase A-S6.
A spectrofluorimetric investigation of the interactions between the subunits of the pancreatic bovine procarboxypeptidase A ternary complex was carried out after covalent insertion of a fluorescent probe at the active center of one of the constituent subunits. The specific insertion of an anthraniloyl group at the active center of subunit II free or bound to subunit I, after its conversion into chymotrypsin II, allowed us to determine the value of the dissociation constant between subunit I and anthraniloyl-chymotrypsin II (Kd = 0.7 +/- 0.1 x 10(-7) M) and between subunit III and the binary complex subunit I-anthraniloyl-chymotrypsin II (Kd = 1.6 +/- 0.3 x 10(-7) M). Moreover, the influence of the association on the flexibility of the active center of chymotrypsin II was deduced from fluorescence polarization measurements and rotational correlation time determination of anthraniloyl-chymotrypsin II free or bound to subunit I. The anthraniloyl group has no motion independently of the whole chymotrypsin II molecule and the binding of subunit I to anthraniloyl-chymotrypsin II results in an increase of the rigidity of the active site in the latter protein.
Granon
S
S
Centre de Biochimie et de Biologie Moléculaire du Centre National de la Recherche Scientifique, Marseille, France.
Kerfelec
B
B
Chapus
C
C
D000818:Animals; D002417:Cattle; D004792:Enzyme Precursors / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D009097:Multienzyme Complexes / Q000302:isolation & purification / Q000378:metabolism*; D010179:Pancreas / Q000201:enzymology; D011485:Protein Binding; D013050:Spectrometry, Fluorescence / Q000379:methods
D004792:Enzyme Precursors; D046911:Macromolecular Substances; D009097:Multienzyme Complexes; C055863:procarboxypeptidase A-S6