Beryllium and aluminum fluorides are good phosphate analogues. These compounds, like orthovanadate, form stable complexes with myosin subfragment 1 (S1) in the presence of MgADP. The formation of the stable S1-nucleotide complexes is characterized by the loss of ATPase activity. For the complete loss of ATPase activity there was necessary a higher concentration of aluminum than of beryllium or vanadate. In the presence of MgATP the onset of the inhibition is delayed, which indicates that stable complexes cannot form when a specific site is occupied by the gamma-phosphate of ATP or by P(i) derived from the gamma-phosphate. The half-lives of the S1-MgADP-(BeF3-), S1-MgADP-(AlF4-), and S1-MgADP-Vi complexes at 0 degrees C are 7, 2, and 4 days, respectively. In the presence of actin the rate of decomposition of all of the complexes is significantly enhanced; however, the order of decomposition is reversed, the fastest rate being observed with beryllium and the slowest with aluminum. The formation of the S1-MgADP-(BeF3-) and S1-MgADP-(AlF4-) complexes is accompanied by an increase in tryptophan fluorescence similar to that observed upon addition of MgATP to S1. The fluorescence increase develops rather slowly, by suggesting that the rate-limiting step in the formation of the stable complex is an isomerization. The rate of the fluorescence change accompanying the formation of the Be complex is faster than that for the Al complex. Addition of vanadate to S1 causes a static quenching of the tryptophan fluorescence.(ABSTRACT TRUNCATED AT 250 WORDS)

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Recent crystallographic studies have suggested structural differences between the complexes of S1 ˙ Mg ˙ ADP with the phosphate analogs aluminium fluoride (AlF−4), vanadate (VO3‐4) and beryllium fluoride (BeFx) [Fisher, A. J., Smith, C. A., Thoden, J. B., Smith, R., Sutoh, K., Holden, H. M. & Rayment, I. (1995) Biochemistry 34, 8960–8972; Smith, R. & Rayment, I. (1996) Biochemistry 35, 5404–54171. In this work, chemical modifications, namely labeling of Cys707 (the reactive SH1 thiol) and Cys707 – Cys697 (SH1‐SH2) cross‐linking, were used to compare the S1 ˙ ADP ˙ BeFx, S1 ˙ ADP ˙ AlF−4 and S1 ˙ ADP ˙ VO3‐4 complexes with specific states of the myosin‐ATPase pathway. Modification of Cys707 with the fluorescent monofunctional reagents 7‐diethylamino‐3‐(4′‐maleimidylphenyl)‐4‐methylcoumarin and N‐iodoacetyl‐N′‐(5‐sulfo‐1‐naphtyl)ethylenediamine has shown that the reactivity of the SH1 group depends on the nucleotide bound to S1. The observed rates of Cys707 modification at 20°C lead to the conclusion that S1˙ ADP ˙ BeFx is similar to S1*˙ ATP, while S1 ˙ ADP ˙ AIF−4 and S1 ˙ ADP ˙ VO43‐ are more similar to S1**˙ ADP ˙ Pi. The conformations of the analog states were also compared by monitoring the dissociation of the fluorescent nucleotide analog 1‐N6‐ethenoadenosine diphosphate (ADP[C2H2]) from the active site of Cys707‐modified (by N‐ethylmaleimide) and Cys707‐Cys697‐cross‐linked (by N,N′‐p‐phenylene dimaleimide) S1 ˙ ADP[C2H2] ˙ AlF−4 and S1 ˙ ADP[C2H2] ˙ BeFx. Our results suggest that the conformations of the S1 ˙ ADP ˙ AlF−4, S1 ˙ ADP ˙ VO3‐4 and S1 ˙ ADP ˙ BeFx, complexes in the Cys707–Cys697 region are distinct from each other, with the former two at least partially resembling the S1**˙ ADP ˙ Pi state, while the latter is similar to the prehydrolyzed S1*˙ ATP state.

Myosin belongs to the family of motor proteins. Its interaction with actin coupled with hydrolysis of ATP is the molecular basis of muscle contraction. The head segment of myosin, called subfragment 1 (S1), contains 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-Pi complex which is accelerated by actin. The substitution of Pi with phosphate analogs (PA), such as vanadate (Vi) or beryllium fluoride (BeF(x)), highly stabilizes the complex. We studied the role of the divalent cations in the ATPase activity and in the formation and decomposition of PA-containing stable complexes by substituting Mg2+ with Fe2+, Mn2+, Ni2+, Co2+, and Ca2+. These metal ions supported the actin activation of S1 ATPase and affected the obtained kinetic parameters, Km 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 formation of the stable ternary S1-MeADP-PA complexes, which cannot be generated in the absence of divalent cations. Upon formation of stable 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 complex 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 Vi- and BeF(x)-containing complexes, respectively. The ATPase activity of S1 is recovered upon addition of actin, which causes the decomposition of the complex. The spontaneous decomposition of the complexes was studied in the presence of ethylenediaminetetraacetic acid (EDTA), which chelates the metal divalent cations released from the complex and prevents its reformation. The rate of decomposition was assessed by monitoring the recovery of the ATPase activity of S1 in the presence of EDTA. The rate of decomposition of the Vi- and BeF(x)-containing complexes follows the order Mn > Fe > Co > Mg > Ni and Ca >> 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 release of PA followed by a rapid dissociation of MeADP from S1. The stability of the complexes has been calculated from the formation and decomposition rates. Except in the case of Mg, the stabilities of the BeF(x) complexes are higher than those containing Vi. The results indicate that the metal cations have a significant role in maintaining the proper structure of the transient state complex in the myosin-catalyzed ATP hydrolysis.

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