Conformational changes around the active site of myosin-ATPase were studied from the reactivity of specific sulfhydryl groups (S
1 and S
2) 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. S
1 and S
2 of myosin reacted with all maleimide derivatives used, but the patterns of change in ATPase activity were different each other. S
1 showed higher reactivity than the SH group of NAC, and the apparent reaction rate to NEM was about 700 times greater than that of S
2. Reagents with aromatic side chain, especially BIPM, showed relatively higher reactivity to S
2 than S
1.
3. S
1 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 S
2 and its susceptibility to nucleotide that S
2 was in buried state.
5. The reactivity of S
1 was increased in the presence of nucleotides, its order being ADP>ATP.
6. From the values of pka of S
1 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 Mg
2+-ATP to the S
1-blocked myosin system did not alter the total amount of thiols reacted with BIPM, but selectively increased that of S
2 to a great extent.
8. Arrhenius plot of the reaction rate of S
2 was biphasic with a bending point around 15°C. In the presence of Mg
2+-ATP, the bending completely disappeared.
On the basis of the results obtained, the state of S
1 and S
2 in the myosin molecule and the flexibility of the conformation of the active site of myosin-ATPase were discussed.
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