COLI F1 F0 -ATP SYNTHASE

F1F0-ATP氧化磷酸化过程中ATP的合成 合酶，占活细胞中合成ATP的大部分； 本研究的目的是了解心绞痛的结构和机制。 尽可能详细地了解F1F0-ATP合酶的作用。这个 将研究大肠杆菌酶，因为它有几个优点，例如它 容易受到分子生物学/基因操纵的影响，它可能是 解离成单独的原生亚单位并重新结合，它可以 以高产量获得。 具体目标是(1)研究催化剂的结构和功能 通过突变、逆转分析和酶学技术获得的位点(2) 研究显著的正催化协作性是至关重要的 用诱变和逆转分析研究该酶的作用机制 (3)从α和β亚单位基因构建嵌合基因，以 嵌合蛋白的表达、纯化及其核苷酸研究 绑定属性，以便区分 腺嘌呤特异的、非催化的α-位点与催化的那些 表征单位本征催化步骤的#-位(4) 正常和突变酶的pH和溶剂效应的催化作用 (5)研究fd质子途径与F1催化剂之间的能量耦合 通过β和β-亚基的突变、逆转分析和 脂多糖诱导核苷酸结合G亲和力变化的检测方法研究进展 和催化现场环境。 三磷酸腺苷驱动的泵在自然界中分布非常广泛，并涉及 在许多疾病状态下；在这里要做的工作将因此具有广泛的 对生物和医学的影响。

Synthesis of ATP during oxidative phosphorylation occurs on the F1F0-ATP synthase enzyme and accounts for the bulk of ATP synthesis in living cells; the goal of this research is to understand the structure and mechanism of action of F1F0-ATP synthase in as much molecular detail as possible. The E. coli enzyme will be studied because of its several advantages e.g. it is readily amenable to molecular biology/genetic manipulation, it may be dissociated into individual native subunits and reassociated, and it may be obtained in high yield. Specific goals are (1) to study structure and function of the catalytic sites by mutagenesis, reversion analysis, and enzymological techniques (2) to study the remarkable positive catalytic cooperativity which is a crucial mechanistic feature of this enzyme by mutagenesis and reversion analysis (3) to construct chimerical genes from alpha and betasubunit genes, to express and purify the chimerical proteins, and to study their nucleotide binding properties in order to distinguish determinant features of the adenine-specific, non-catalytic alpha-sites from those of the catalytic #-sites (4) to characterize intrinsic catalytic steps of single-site catalysis in terms of pH and solvent effects in normal and mutant enzymes (5) to study energy coupling between the FD proton-pathway and F1 catalytic sites by mutagenesis of beta and &-subunits, reversion analysis, and development of assays of Lpinduced changes in nucleotide-binding g affinity and catalytic site environment. 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.