EFFECT OF DIVALENT-CATIONS ON THE FORMATION AND STABILITY OF MYOSIN SUBFRAGMENT 1-ADP-PHOSPHATE ANALOG COMPLEXES

Myosin belongs to the family of motor proteins, Its interaction with a ctin coupled with hydrolysis of ATP is the molecular basis of muscle c ontraction. The head segment of myosin, called subfragment 1 (S1), con tains the distinct binding sites for ATP and actin and responsible for the ATPase activity. The rate-limiting step of the ATP hydrolysis is the dissociation of the S1 . MgADP . P-i complex which is accelerated by actin. The substitution of Pi with phosphate analogs (PA), such as vanadate (Vi) or beryllium fluoride (BeFx), highly stabilizes the comp lex, We studied the role of the divalent cations in the ATPase activit y and in the formation and decomposition of PA-containing stable compl exes by substituting Mg2+ with Fe2+, Mn2+, Ni2+, Co2+, and Ca2+. These metal ions supported the actin activation of S1 ATPase and affected t he obtained kinetic parameters, K-M and V-max. The ATPase activity of S1 in the absence of actin increased with the increasing ionic radius of the metal (Me) ions, These ions also substituted for Mg2+ in the fo rmation of the stable ternary S1 . MeADP . PA complexes, which cannot be generated in the absence of divalent cations, Upon formation of sta ble ternary complexes, S1 reversibly loses its ability to catalyze the hydrolysis of ATP, The formation of the complexes can be followed by monitoring the disappearance of the ATPase activity. The rate of the c omplex formation depends on the divalent cation present and decreases in the order Mn > Fe > Ni > Co > Mg and Ca, Mn > Fe > Mg > Co in the V i- and BeFx-containing complexes, respectively. The ATPase activity of S1 is recovered upon addition of actin, which causes the decompositio n of the complex. The spontaneous decomposition of the complexes was s tudied in the presence of ethylenediaminetetraacetic acid (EDTA), whic h chelates the metal divalent cations released from the complex and pr events its reformation. The rate of decomposition was assessed by moni toring the recovery of the ATPase activity of S1 in the presence of ED TA. The rate of decomposition of the Vi- and BeFx-containing complexes follows the order Mn > Fe > Co > Mg > Ni and Ca much greater than Mn > Fe > Co > Mg, respectively. The rate of decomposition increases with the increasing ionic radius of the metal ions, similarly as observed in the case of ionic radius dependence of the ATPase activity, On the basis of this similarity, it is assumed that the decomposition of the complexes consists of two steps, the first step being the very slow re lease of PA followed by a rapid dissociation of MeADP from S1. The sta bility of the complexes has been calculated from the formation and dec omposition rates. Except in the case of Mg, the stabilities of the BeF , complexes are higher than those containing Vi. The results indicate that the metal cations have a significant role in maintaining the prop er structure of the transient state complex in the myosin-catalyzed AT P hydrolysis.