Structure and Function of the Two Heads of the Myosin Molecule

The kinetic properties of the hydrolyses of 8-Br ATP and 8-SCH³ ATP by myosin [EC 3.6.1.3] and actomyosin were compared with those of ATP, and the following results were obtained. The Ca-NTPase activities of myosin using these two ATP analogs as substrates were smaller than that of ATPase, and the NTPase activities toward these analogs were strongly suppressed by EDTA. The Mg-NTPase activities toward these analogs were higher in a medium of high ionic strength than in a medium of low ionic strength, in contrast to the activity of Mg-ATPase. These analogs did not produce any initial burst of P₁ liberation, activation of myosin NTPase by F-actin, or superprecipitation of actomyosin. The interactions between 8-Br ATP and HMM, acto-HMM, actomyosin, and myofibrils were studied in detail in the presence of Mg²⁺ in medium of low ionic strength. The Michaelis constant, K_m, and the maximum rate, V_m, of 8-Br ATPase of HMM were 27 μM and 21 min⁻¹, respectively. The fluorescence change of HMM induced by 8-Br ATP also followed the Michaelis-Menten equation, and the Michaelis constant, K_f1, was as low as 4 μM. Acto-HMM and acto-S-1 were fully dissociated by the addition of 8-Br ATP. The relation between the extent of dissociation of acto-HMM and the concentration of 8-Br ATP followed the Michaelis-Menten equation, and the apparent dissociation constant, K_d, was 22 μm. This K_d value is almost equal to the K_m value of 8-Br ATPase of HMM described above. Myofibrillar contraction was not supported by 8-Br ATP. It was concluded that in the myosin NTPase reaction with 8-Br ATP as a substrate, M₂NTP but not $MPNDP$ is formed in route (1), while $MNTP$ is formed in route (2). It was also concluded that the key intermediate for the actomyosin NTPase reaction is $MPNDP$, and the dissociation of acto-HMM is induced by the formation of M₂NTP and $MNTP$ in routes (1) and (2), respectively.