Thiols of Myosin: IV. “Abnormal” Reactivity of S1 Thiol and the Conformational Changes around S2 Thiol1

Abstract

The flexibility of the tertiary structure around the active site of myosin ATPase [EC 3. 6. 1. 3] was studied using the reactivity of two specific thiol groups, S₁ and S₂, as a structural probe. The following four maleimide derivatives were used as thioldirected reagents: N-ethylmaleimide (NEM), N-(4-methoxy-2-benzimidazolyl methyl) maleimide (MBM), N-(p-(2-benzimidazolyl)phenyl)maleimide (BIPM) and N-(4-dimethylamino-3, 5-dinitrophenyl)maleimide (DDPM). 1. All the maleimide derivatives used activated the Ca²⁺-ATPase activity and inhibited the EDTA-ATPase activity, like NEM, indicating that they modified S₁. The rate of modification of S₁ by NEM and BIPM increased with increasing pH, while that by DDPM decreased. BIPM simultaneously modified S₁ and S₂. 2. S₁ showed much higher reactivity toward the maleimides, except for BIPM, than did N-acetylcysteine (N-Ac-Cys) a low-molecular-weight model compound. The extremely small pKa value of S₁, 6.28, accounted for this high reactivity. In addition, the ATP-induced increase in its reactivity indicated that S₁ was in a buried state. Kinetic analysis showed that the tertiary structure around S₁ at alkaline pH differed from that at acidic pH. 3. The apparent rate constant of S₂-modification with NEM was approximately one seven-hundredth and one four-hundredth of those of S₁ and N-Ac-Cys, respectively. Fluorimetric studies using BIPM revealed that S₂ in the buried state was exposed upon adding ATP; this was compensated by the burying of some other thiol group(s) (Sp). Non-linearity of the Arrhenius plots of the reaction rate of S₂ suggested that the S₂ region of myosin had different conformations at high and low temperatures, the transition temperature being 10–15°. This non-linearity completely disappeared in the presence of Mg²⁺-ATP. On the other hand, Arrhenius plots for the thiols reactive to BIPM did not show non-linearity in the presence or absence of ATP.