Murataliev M B, Boyer P D
Molecular Biology Institute, University of California Los Angeles 90024-1570.
Eur J Biochem. 1992 Oct 15;209(2):681-7. doi: 10.1111/j.1432-1033.1992.tb17336.x.
The presence of ATP at non-catalytic sites of the chloroplast F1-ATPase (CF1) eliminates a considerable lag in onset of enzyme activity that otherwise occurs in the presence of bicarbonate [Milgrom, Y. M., Ehler, L. & Boyer, P. D. (1991) J. Biol. Chem. 266, 11551-11558]. Sulfite is known to be much more effective than bicarbonate in stimulating ATPase activity CF1. Results reported here show that when assayed in the presence of sulfite, CF1, with some non-catalytic sites empty or filled with GT(D)P, is able to hydrolyze both ATP and GTP. Thus, the presence of adenine nucleotides at non-catalytic sites is not necessary for catalytic turnover of CF1. However, even though CF1 with empty non-catalytic sites shows a significant initial activity, the prior binding of adenine nucleotides at non-catalytic site(s) results in further activation of MgATPase and MgGTPase activities, even at relatively high sulfite and substrate concentrations. Although extensive activation of CF1 results from the presence of sulfite, with or without nucleotide binding at non-catalytic sites, the Km remains constant, at about 50 microM for MgATP and 400 microM for MgGTP. The results obtained show that the ATPase activity of CF1 is determined by the fraction of the active enzyme. The inactive CF1.ADP.Mg2+ formed during MgATP hydrolysis can be rapidly trapped by azide to provide a measure of the fraction of inactive enzyme. Increasing the concentration of sulfite increases the fraction of active CF1 in the assay medium. Measurements with radioactively labeled nucleotides show that the presence of ATP at non-catalytic sites promotes the ATP-dependent release of inhibitory ADP from a catalytic site. The activating effect of ATP binding at non-catalytic sites results from increasing the portion of CF1 in an active state during steady-state ATP hydrolysis.
叶绿体F1 - ATP酶（CF1）非催化位点上ATP的存在消除了酶活性起始阶段相当长的延迟，否则在碳酸氢盐存在的情况下会出现这种延迟[米尔格罗姆，Y. M.，埃勒，L. & 博耶，P. D.（1991年）《生物化学杂志》266，11551 - 11558]。已知亚硫酸盐在刺激CF1的ATP酶活性方面比碳酸氢盐有效得多。此处报道的结果表明，当在亚硫酸盐存在下进行测定时，一些非催化位点为空或填充有GT（D）P的CF1能够水解ATP和GTP。因此，非催化位点上腺嘌呤核苷酸的存在对于CF1的催化周转并非必要。然而，尽管非催化位点为空的CF1显示出显著的初始活性，但腺嘌呤核苷酸在非催化位点的预先结合会导致MgATP酶和MgGTP酶活性进一步激活，即使在相对较高的亚硫酸盐和底物浓度下也是如此。尽管亚硫酸盐的存在会导致CF1广泛激活，无论非催化位点有无核苷酸结合，Km保持恒定，MgATP约为50微摩尔，MgGTP约为400微摩尔。所获得的结果表明，CF1的ATP酶活性由活性酶的比例决定。MgATP水解过程中形成的无活性CF1·ADP·Mg2 + 可被叠氮化物迅速捕获，以提供无活性酶比例的一种测量方法。增加亚硫酸盐的浓度会增加测定介质中活性CF1的比例。用放射性标记核苷酸进行的测量表明，非催化位点上ATP的存在促进了抑制性ADP从催化位点的ATP依赖性释放。非催化位点上ATP结合的激活作用源于在稳态ATP水解过程中增加处于活性状态的CF1的比例。
