Heterogeneous flexibilities of the active site domains of homodimeric creatine kinase: effect of substrate.
(1) A single subunit and both subunits of creatine kinase from rabbit muscle was derivatized at the active site with the thiol-specific reagent 2-(4'-(iodoacetamido)anilino)-naphthalene-6-sulfonic acid. (2) The highly biphasic kinetics of the labelling reaction were characterized from measurements of activity, steady-state fluorescence and anisotropy. Derivatization of one thiol and both thiols resulted in 48 and 100% inhibition, respectively. The dead-end complex (DEC), consisting of creatine, MgADP and protein, inhibited the rate, but not the extent, of derivatization and resulted in a 2-fold increase in fluorescence. (3) The fluorescence of singlylabelled (1AANS/CK) and doublylabelled (2AANS/CK) protein exhibited three discrete lifetime components or a two-term Lorentzian distribution. The decay laws for both preparations were not remarkably different, except that, unlike 1AANS/CK, the longer decay component of 2AANS/CK was distributed, which narrowed in the presence of the DEC. (4) The steady-state anisotropies of 1AANS/CK and 2AANS/CK at 25 degrees C were 0.305 and 0.240, respectively. It was concluded that the fast reacting site was immobile and the slow reacting site was flexible. Kinetics of labelling and anisotropy emission spectra indicated that the DEC immobilized the flexible site. (5) The anisotropy decay of 1AANS/CK with and without the DEC was described by a rotational correlation time of about 50 ns, characteristic of the molecular rotation of the CK dimer. At least two terms were required to fit the data for 2AANS/CK, indicating additional segmental motion which was eliminated upon formation of the DEC. (6) Energy transfer from tryptophans to AANS indicated movement of approx. 3 A accompanying formation of the DEC.
Grossman
S H
SH
Department of Chemistry, University of South Florida, Tampa 33620.
France
R M
RM
Mattheis
J R
JR
D000244:Adenosine Diphosphate / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D003401:Creatine / Q000737:chemistry; D003402:Creatine Kinase / Q000737:chemistry*; D066298:In Vitro Techniques; D008024:Ligands; D009038:Motion; D009132:Muscles / Q000201:enzymology; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry
D008024:Ligands; D000244:Adenosine Diphosphate; D014364:Tryptophan; D003402:Creatine Kinase; D003401:Creatine