Takenaka H, Ikehara M, Tonomura Y
J Biochem. 1976 Dec;80(6):1381-92. doi: 10.1093/oxfordjournals.jbchem.a131411.
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 Pi 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, Km, and the maximum rate, Vm, of 8-Br ATPase of HMM were 27 muM 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, Kf1, was as low as 4 muM. 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, Kd, was 22 muM. This Kd value is almost equal to the Km 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 MNDPP 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 MNDPP, and that dissociation of acto-HMM is induced by the formation of M2NTP and MNTP in routes (1) and (2), respectively.
将肌球蛋白[EC 3.6.1.3]和肌动球蛋白对8 - Br ATP及8 - SCH₃ ATP的水解动力学性质与ATP的水解动力学性质进行了比较，得到以下结果。以这两种ATP类似物为底物时，肌球蛋白的Ca - NTP酶活性低于ATP酶活性，且EDTA能强烈抑制其对这些类似物的NTP酶活性。与Mg - ATP酶活性相反，在高离子强度介质中，肌球蛋白对这些类似物的Mg - NTP酶活性高于低离子强度介质中的活性。这些类似物不会引发Pi释放的初始爆发、F - 肌动蛋白对肌球蛋白NTP酶的激活或肌动球蛋白的超沉淀。在低离子强度介质中Mg²⁺存在的情况下，详细研究了8 - Br ATP与重酶解肌球蛋白（HMM）、肌动蛋白 - 重酶解肌球蛋白（acto - HMM）、肌动球蛋白及肌原纤维之间的相互作用。HMM的8 - Br ATP酶的米氏常数（Km）和最大反应速率（Vm）分别为27 μM和21 min⁻¹。8 - Br ATP诱导的HMM荧光变化也遵循米氏方程，其米氏常数（Kf1）低至4 μM。添加8 - Br ATP可使肌动蛋白 - 重酶解肌球蛋白（acto - HMM）和肌动蛋白 - S1（acto - S - 1）完全解离。acto - HMM的解离程度与8 - Br ATP浓度之间的关系遵循米氏方程，表观解离常数（Kd）为22 μM。该Kd值几乎等于上述HMM的8 - Br ATP酶的Km值。8 - Br ATP不能支持肌原纤维收缩。得出结论：在以8 - Br ATP为底物的肌球蛋白NTP酶反应中，途径（1）中形成的是M₂NTP而非MNDPP，而途径（2）中形成的是MNTP。还得出结论：肌动球蛋白NTP酶反应的关键中间体是MNDPP，并且acto - HMM的解离分别由途径（1）和（2）中M₂NTP和MNTP的形成所诱导。
