Abstract
Conformational changes around the active site of myosin-ATPase were studied from the reactivity of specific sulfhydryl groups (S1 and S2) to maleimide derivatives, 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. Apparent second order rate constants of the reaction of maleimide derivatives to a low molecular model compound, N-acethylcysteine (NAC), were BIPM>>NEM>>DDPM>MBM in order.
2. S1 and S2 of myosin reacted with all maleimide derivatives used, but the patterns of change in ATPase activity were different each other. S1 showed higher reactivity than the SH group of NAC, and the apparent reaction rate to NEM was about 700 times greater than that of S2. Reagents with aromatic side chain, especially BIPM, showed relatively higher reactivity to S2 than S1.
3. S1 has “abnormal” dissociation constant, 6.28 as the pka value, as compared to that of cysteine thiol, higher than 9.0.
4. It was confirmed from the reactivity of S2 and its susceptibility to nucleotide that S2 was in buried state.
5. The reactivity of S1 was increased in the presence of nucleotides, its order being ADP>ATP.
6. From the values of pka of S1 the active site of myosin was deduced to have different conformations depending on pH with the critical one around 7.0.
7. The addition of Mg2+-ATP to the S1-blocked myosin system did not alter the total amount of thiols reacted with BIPM, but selectively increased that of S2 to a great extent.
8. Arrhenius plot of the reaction rate of S2 was biphasic with a bending point around 15°C. In the presence of Mg2+-ATP, the bending completely disappeared.
On the basis of the results obtained, the state of S1 and S2 in the myosin molecule and the flexibility of the conformation of the active site of myosin-ATPase were discussed.
