CHARACTERIZATION OF E.COLI F1FO-ATP SYNTHASE

氧化磷酸化过程中ATP的合成发生在F1Fo-ATP合成酶上，并且是活细胞中ATP合成的主要部分。ATP合成酶是一种特殊的酶，因为它作为分子马达，跨膜质子梯度的能量通过亚基旋转耦合在三个不对称但相互转换的催化位点合成ATP。反过来，ATP水解驱动亚基旋转和质子泵送。本研究的长期目标是尽可能详细地了解F1Fo-ATP合酶的分子机制。大肠杆菌酶具有许多优点，如易于进行分子生物学/遗传学操作，可以快速高产地获得，并且可以在具有优异ATP合成活性的脂质体中重组。具体目标是(1)确定ATP合成过程中质子梯度存在下F1Fo的催化位点占用和核苷酸结合参数；(2)通过诱变γ中面对α和β的残基，鉴定γ / α和γ / β亚基之间的功能相互作用；(3)阐明在ATP水解过程中，通过过渡态到ADP基态的形成和崩溃，催化α / β亚基界面产生的蛋白质运动；(4)表征低ATP浓度下的ATP水解，其中120摄氏度的转子步进；(5)定子茎的遗传分析，从本实验室先前显示的α和δ亚基突变开始，中断F1与Fo的结合和能量耦合。atp驱动的泵在自然界中分布非常广泛，并与许多疾病状态有关。因此，这里所做的工作将对生物学和医学产生广泛的影响。

Synthesis of ATP during oxidative phosphorylation occurs on the F1Fo-ATP synthase enzyme and accounts of the bulk of ATP synthesis in living cells. ATP synthase is an extraordinary enzyme because it acts as a molecular motor the energy of the transmembrane proton gradient is coupled through subunit rotation to the synthesis of ATP in three asymmetric but interconverting catalytic sites. In reverse, ATP hydrolysis drives subunit rotation and proton pumping. The long-term goal of this research is to understand the mechanism of F1Fo-ATP synthase in as much molecular detail as possible. The E. coli enzyme will be used because of its many advantages, e.g. it is readily amenable to molecular biology/genetic manipulations, it may be rapidly obtained in high yield, and it may be reconstituted in liposomes with excellent ATP synthesis activity. Specific goals are (1) determination of catalytic sites occupancy and nucleotide binding parameters in F1Fo in presence of a proton gradient, during ATP synthesis; (2) identification of functional interactions between gamma/alpha and gamma/beta subunits, by mutagenesis of residues in gamma which face alpha and beta; (3) elucidation of protein movements generated at the catalytic alpha/beta subunit interface as ATP hydrolysis proceeds through formation and collapse of the transition state to the ADP ground- state; (4) characterization of ATP hydrolysis at low ATP concentrations, where 120 degrees Celsius stepping of the rotor is seen; and (5) genetic analysis of the stator stalk, starting from mutations in alpha and delta subunits shown previously in this laboratory to interrupt both F1 binding to Fo and energy coupling. ATP-driven pumps are very widely distributed in nature, and are involved in many disease states. Work to be done here will consequently have broad impact in biology and medicine.