FORMATION OF STABLE INHIBITORY COMPLEXES OF MYOSIN SUBFRAGMENT-1 USING FLUOROSCANDIUM ANIONS

Evidence is presented that MgADP can be noncovalently trapped in myosi n subfragment 1 in the presence of ScFx resulting in the concomitant l oss of ATPase function. The rate of inactivation in the presence of Mg Cl2 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 isomerizatio n step is responsible for formation of a stable ternary complex, S1 . MgADP . ScFx in a manner analogous to that proposed for the Vi stabili zed 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 subf ragment 1 in the absence of MgCl2 indicating the formation of an S1 . ADP . ScFx complex. The stability of these complexes at 4 degrees C wa s studied by following the loss of trapped [C-14]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 disti nct states as previously proposed for the ternary complex stabilized b y Vi (Mihashi, K., Ooi, A., and Hiratsuka, T. (1990) J. Biochem. (Toky o) 107, 464-469). Formation of these complexes resulted in a marked en hancement of the intrinsic tryptophyl fluorescence suggesting that con formationally they may resemble the steady-state intermediate formed w ith 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 cleava ge reactions and that it is not displaced by the added Vi.