Kinetic and equilibrium analysis of the interactions of actomyosin subfragment-1.ADP with beryllium fluoride.
The hypothesis that the stable ternary complex formed between myosin subfragment-1, MgADP and beryllium fluoride (BeF3-), denoted S-1 not equal to .ADP.BeF3-, is an analog of the intermediate state S-1**.ADP.P(i) has been tested in this work by examining the interactions of S-1 not equal to .ADP.BeF3- with actin. Equilibrium binding measurements revealed that actin bound weakly to the S-1 not equal to .ADP.BeF3- complex (Ka = 10(4) M-1) in the presence of 40 mM KCl. The stability of this complex was strongly salt-dependent. The association constant of BeF3- to the acto-S-1.ADP complex (KBe approximately 10(3) M-1) was 100-fold weaker than its binding to the S-1.ADP complex. While inhibiting the S-1 ATPase strongly, BeF3- had no effect on the Vmax value (10 +/- 1.0 s-1) of the actin-activated ATPase of S-1. The rates of BeF3- binding and dissociation from the acto-S-1.ADP.BeF3- complex were determined by stopped-flow measurements. The hyperbolic dependence of the rates of BeF3- binding to acto-S-1.ADP (kobs) on BeF3- concentrations suggested that the acto-S-1.ADP.BeF3- complex was formed in at least two steps: binding followed by isomerization. The binding constant was 1.2 x 10(3) M-1, and the maximum kobs was 2.5 s-1. The dissociation of BeF3- from the acto-S-1.ADP.BeF3- complex was monitored via decrease in the fluorescence of 1-N6-ethenoadenosine diphosphate (epsilon ADP). The fluorescence decrease fitted two exponential terms.(ABSTRACT TRUNCATED AT 250 WORDS)
Phan
B C
BC
Department of Chemistry and Biochemistry, University of California, Los Angeles 90024.
Faller
L D
LD
Reisler
E
E
D000199:Actins / Q000737:chemistry; D000244:Adenosine Diphosphate / Q000737:chemistry*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D001608:Beryllium / Q000737:chemistry*; D004789:Enzyme Activation; D005459:Fluorides / Q000737:chemistry*; D007700:Kinetics; D015879:Myosin Subfragments / Q000737:chemistry*; D011817:Rabbits
D000199:Actins; D015879:Myosin Subfragments; C035982:beryllium fluoride; D000244:Adenosine Diphosphate; D000251:Adenosine Triphosphatases; D001608:Beryllium; D005459:Fluorides