Formation of stable inhibitory complexes of myosin subfragment 1 using fluoroscandium anions.
Evidence is presented that MgADP can be noncovalently trapped in myosin subfragment 1 in the presence of ScFx resulting in the concomitant loss of ATPase function. The rate of inactivation in the presence of MgCl2 at 25 degrees C is 8.7 M-1 s-1 which is too slow for a simple collisional mechanism and suggests that a subsequent slow isomerization step is responsible for formation of a stable tenary complex, S1.MgADP.ScFx in a manner analogous to that proposed for the Vi stabilized complex by Goodno (Goodno, C. C. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 2620-2624). It is also found that ADP can be trapped in subfragment 1 in the absence of MgCl2 indicating the formation of an S1.ADP.ScFx complex. The stability of these complexes at 4 degrees C was studied by following the loss of trapped [14C]ADP with a chase with ADP. The rate of nucleotide loss at 4 degrees C was biphasic for both complexes suggesting that the inhibitory complexes exist in two distinct states as previously proposed for the ternary complex stabilized by Vi (Mihashi, K., Ooi, A., and Hiratsuka, T. (1990) J. Biochem. (Tokyo) 107, 464-469). Formation of these complexes resulted in a marked enhancement of the intrinsic tryptophyl fluorescence suggesting that conformationally they may resemble the steady-state intermediate formed with MgATP. The failure to observe photolysis in the presence of excess Vi at sites associated with the ATP consensus sequence suggests that in these complexes ScFx occupies the site responsible for these cleavage reactions and that it is not displaced by the added Vi.
Gopal
D
D
Department of Biology, College of Arts and Sciences, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Burke
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M
D000199:Actins / Q000737:chemistry; D000244:Adenosine Diphosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D017301:Ca(2+) Mg(2+)-ATPase / Q000037:antagonists & inhibitors; D005459:Fluorides / Q000737:chemistry*; D007700:Kinetics; D015879:Myosin Subfragments / Q000737:chemistry*; D012538:Scandium / Q000737:chemistry*
D000199:Actins; D015879:Myosin Subfragments; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D017301:Ca(2+) Mg(2+)-ATPase; D005459:Fluorides; D012538:Scandium