J. Biol. Chem., Vol. 265, Issue 18, 10383-10388, 06, 1990
Spectrofluorimetric investigation of the interactions between the subunits of bovine pancreatic procarboxypeptidase A-S6
S Granon, B Kerfelec and C Chapus
Centre de Biochimie et de Biologie Moleculaire du Centre National de la Recherche Scientifique, Marseille, France.
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.
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