Bagshow C R, Reed G H
J Biol Chem. 1976 Apr 10;251(7):1975-83.
Electron paramagnetic resonance spectroscopy and water proton relaxation rate (PRR) measurements were used to characterize a complex formed at the myosin subfragment 1 (S1) ATPase site with stoichiometric amounts of Mn(II) and ADP. In the absence of nucleotide, Mn(II) binding at the active site is very weak, although two other classes of sites for Mn(II) on subfragment 1 were identified which are not directly involved in the ATPase reaction. A high affinity Mn(II) site (termed L-site with KL = 3 muM) is associated with a region of the molecule which is susceptible to proteolysis (probably the LC2 light chain subunit) since its stoichiometry depends on the conditions employed for the preparation of subfragment 1 during the papain treatment of myosin. In addition there are a number of weak sites for Mn(II) (termed N-sites) probably associated with anionic groups on the surface of the molecule. In order to study the properties of Mn(II) and ADP bound at the active site by magnetic resonance techniques, subfragment 1 preparations virtually free of the L-site were used, since such an ancillary site competes for the available Mn(II). MnADP binds to subfragment 1 with an apparent dissociation constant, KT, of about 4 muM at 25 degrees. The resultant complex, S1-MnADP, has a low PRR enhancement factor (1.7 at 24.3 MHZ), and its frequency (magnetic field) dependence indicates that this is because there are no readily exchangeable water molecules within the first coordination sphere of Mn(II. Relaxation of the bulk solvent is mediated by protons bound transiently within the outer spheres (4 to 7 A) of the Mn(II). A nitroxide spin label attached to the reactive thiol group of subfragment 1 enhances the solvent PRR, and this property is sensitive to the binding of MgADP to the active site. However, no dipolar spin-spin interaction was detected between the nitroxide group and Mn(II) in the S1-MnADP complex, indicating that the metal ion and thiol group are well separated.
电子顺磁共振光谱法和水质子弛豫率（PRR）测量被用于表征在肌球蛋白亚片段1（S1）的ATP酶位点与化学计量的锰（II）和ADP形成的复合物。在没有核苷酸的情况下，锰（II）在活性位点的结合非常弱，尽管在亚片段1上鉴定出了另外两类锰（II）的结合位点，它们不直接参与ATP酶反应。一个高亲和力的锰（II）位点（称为L位点，KL = 3 μM）与分子中一个易受蛋白水解作用的区域（可能是LC2轻链亚基）相关，因为其化学计量取决于在木瓜蛋白酶处理肌球蛋白过程中制备亚片段1所采用的条件。此外，还有一些锰（II）的弱结合位点（称为N位点），可能与分子表面的阴离子基团有关。为了通过磁共振技术研究结合在活性位点的锰（II）和ADP的性质，使用了几乎不含L位点的亚片段1制剂，因为这样一个辅助位点会竞争可用的锰（II）。在25℃时，锰 - ADP以约4 μM的表观解离常数KT与亚片段1结合。形成的复合物S1 - MnADP具有低的PRR增强因子（在24.3 MHz时为1.7），其频率（磁场）依赖性表明这是因为在锰（II）的第一配位球内没有易于交换的水分子。大量溶剂的弛豫是由在锰（II）的外层球（4至7 Å）内短暂结合的质子介导的。连接到亚片段1的反应性巯基上的氮氧化物自旋标记增强了溶剂的PRR，并且这种性质对MgADP与活性位点的结合敏感。然而，在S1 - MnADP复合物中未检测到氮氧化物基团与锰（II）之间的偶极自旋 - 自旋相互作用，这表明金属离子和巯基基团相距较远。
