STRUCTURE AND MECHANISM OF F0F1-ATP SYNTHASES

F0F1-ATP 合成酶的结构和机制

基本信息

批准号：
2444462
负责人：
RICHARD L CROSS
金额：
$ 28.79万
依托单位：
UPSTATE MEDICAL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1976
资助国家：
美国
起止时间：
1976-05-01 至 1998-06-30
项目状态：
已结题

项目摘要

ATP synthesis by oxidative phosphorylation is catalyzed by F0F1-ATP synthases found embedded in mitochondrial and bacterial membranes. The F1 portion of the complex contains the catalytic sites, can be readily solubilized, and has a subunit stoichiometry of alpha3-beta3-gamma-delta- epsilon. The structure, mechanism, and regulation of F1 is the subject of this proposal. F1 contains three catalytic and three noncatalytic nucleotide-binding sites. A topological model for the sites has been constructed based on affinity labeling, mutagenesis, predictions of secondary structure, and comparisons with proteins of known structure. The model places sites at or near alpha/beta interfaces, where a pair of catalytic and noncatalytic sites are oriented like the ATP and AMP sites of adenylate kinase. Residues predicted to interact with bound nucleotides will be altered by site-specific mutagenesis of the E. coli enzyme. The role of each will be assessed by kinetic analysis and nucleotide binding measurements. In some cases, where residues in the putative adenine-binding subdomains are changed to tyrosine, it may be possible to obtain a direct confirmation of their presence by testing for reactivity towards 2-azido-ATP. Similarly, when substituting lysine for residues thought to interact with phosphoryl groups, reactivity towards PLP-AMP, PLP-ADP, and PLP-ATP will be tested. Primary mutations that allow assembly of the synthase but give the unc- phenotype (low or no growth on succinate) will be used to select for second-site suppressor mutations. A targeted approach for random mutagenesis will employ double-stranded cassettes for small regions and a modified polymerase chain reaction for larger segments. Particular emphasis will be given to the identification of interacting sites between the alpha and beta subunits. Novel bifunctional probes will be used to further examine the orientation of sites. Bis-(2-azido)APxA will be tested for its ability to crosslink tyrosine that are known to be present at catalytic and noncatalytic sites. PLP-(2-azido)ADP will be used to establish the location of an alpha- subunit lysyl residue. Experiments are designed to determine whether noncatalytic site-bound nucleotides can act as acid catalysts or participate in the slow transfer of phosphoryl groups to and from catalytic sites. Further studies of the mechanism will include attempts to determine whether the three catalytic sites turn over in a sequential manner and whether rotary motion is obligatory in coupled reactions. Improvements in the kinetic analysis of mutant EcF1 will include determining conditions that avoid inhibition by the epsilon-subunit and by MgADP.

通过氧化磷酸化的ATP合成由F0F1-ATP催化合成酶发现在线粒体和细菌膜中。 F1 该综合体的一部分包含催化位点，可以很容易地溶解化，并具有alpha3-beta3-gamma-delta-的亚基化学计量法 Epsilon。 F1的结构，机制和调节是这个建议。 F1包含三个催化和三个非催化核苷酸结合站点。基于该站点的拓扑模型是基于亲和力标记，诱变，二级结构的预测和与已知结构的蛋白质进行比较。模型位置位于或靠近alpha/beta界面，一对催化和非催化像腺苷酸激酶的ATP和AMP位点一样定向位置。预计将与结合核苷酸相互作用的残基将改变大肠杆菌酶的位点特异性诱变。每个的作用将是通过动力学分析和核苷酸结合测量评估。在某些人中情况，假定的腺嘌呤结合子域中的残留物为更改为酪氨酸，可能有可能直接确认通过测试对2-齐多ATP的反应性，它们的存在。相似地，将赖氨酸代替被认为与磷酸相互作用的残基时将测试组，对PLP-AMP，PLP-ADP和PLP-ATP的反应性。允许合成酶组装但给出UNC-的主要突变表型（琥珀酸酯上的低或没有生长）将用于选择第二位抑制剂突变。随机的目标方法诱变将用于小区域的双链盒和改良的聚合酶链反应，用于较大的片段。特别的将重点放在识别互动站点之间的识别 alpha和beta亚基。新型双功能探针将用于进一步检查方向网站。 BIS-（2-齐多）APXA的交联能力将进行测试已知存在于催化和非催化部位的酪氨酸。 PLP-（2-齐多）ADP将用于建立α-的位置亚基赖氨基残基。实验旨在确定是否非催化部位结合的核苷酸可以充当酸催化剂或参与磷酸组往返磷酸基团的缓慢转移催化位点。对该机制的进一步研究将包括尝试确定这三个催化位点是否以顺序翻转和旋转运动是否在耦合反应中是必不可少的。改进突变ECF1的动力学分析将包括确定条件避免了Epsilon-Subunit和MGADP的抑制作用。

项目成果

期刊论文数量（39）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Rapid hydrolysis of ATP by mitochondrial F1-ATPase correlates with the filling of the second of three catalytic sites.

线粒体 F1-ATP 酶对 ATP 的快速水解与三个催化位点中第二个的填充相关。

DOI：
10.1073/pnas.0507139102
发表时间：
2005
期刊：
Proceedings of the National Academy of Sciences of the United States of America.
影响因子：
0
作者：
Milgrom,YakovM;Cross,RichardL
通讯作者：
Cross,RichardL

Bi-site activation occurs with the native and nucleotide-depleted mitochondrial F1-ATPase.

双位点激活发生在天然和核苷酸耗尽的线粒体 F1-ATP 酶上。