Melese T, Xue Z X, Stempel K E, Boyer P D
Molecular Biology Institute, University of California, Los Angeles 90024-1570.
J Biol Chem. 1988 Apr 25;263(12):5833-40.
When heat-activated F1-ATPase from chloroplasts was repeatedly exposed to Mg2+ and 2-azido-ATP, followed by separation from medium nucleotides and photolysis, a total of two sites per enzyme, both catalytic and noncatalytic, were labeled. In a coupled assay with pyruvate kinase about half the activity was lost when one site per enzyme was modified. However, increased modification resulted in no further loss of activity. In contrast, methanol-sulfite activation of the enzyme showed a loss of most of the catalytic capacity when one site per enzyme was modified. Predominant labeling of either one catalytic or one noncatalytic site caused a loss of most of the activity in either assay. An indication that the enzyme modified at one site retained some catalytic activity was verified by measurement of the [18O]Pi species formed when [gamma-18O]ATP was hydrolyzed by partially derivatized enzyme. With either catalytic or noncatalytic site modification, the distributions of [18O]Pi species formed showed that the modified enzyme had different catalytic characteristics. An interpretation is that with modification by azido nucleotides at either catalytic or noncatalytic sites, capacity for rapid catalysis is largely lost but the remaining sites retain weak modified catalytic properties.
当叶绿体中热激活的F1 - ATP酶反复暴露于Mg2 +和2 - 叠氮基 - ATP，随后与培养基中的核苷酸分离并进行光解时，每个酶总共两个位点（催化位点和非催化位点）被标记。在与丙酮酸激酶的偶联测定中，当每个酶的一个位点被修饰时，约一半的活性丧失。然而，修饰增加并未导致活性进一步丧失。相比之下，该酶的甲醇 - 亚硫酸盐激活显示，当每个酶的一个位点被修饰时，大部分催化能力丧失。在任何一种测定中，一个催化位点或一个非催化位点的主要标记都会导致大部分活性丧失。通过测量部分衍生化的酶水解[γ - 18O]ATP时形成的[18O]Pi种类，证实了在一个位点修饰的酶保留了一些催化活性这一迹象。无论是催化位点还是非催化位点被修饰，形成的[18O]Pi种类的分布表明，修饰后的酶具有不同的催化特性。一种解释是，在催化位点或非催化位点被叠氮核苷酸修饰后，快速催化能力基本丧失，但其余位点保留了较弱的修饰催化特性。
