STRUCTURE/FUNCTION OF THE E COLI H+ ATPASE

点击翻译按钮获取中文摘要

DESCRIPTION: One of the central problems in modern biology is the interconversion of different forms of biological energy during the synthesis of ATP. F1Fo ATPases synthesize most of the ATP in living systems. They have the same basic structure and function in mitochondria, chloroplasts, and bacteria. Bacterial ATPases, especially the E. coli enzyme, have proven to be valuable systems for understanding the structure and function of these enzymes. The proposed research addresses how the energy of proton movement is coupled to ATP synthesis or hydrolysis. Specifically, energy-coupling involving the C-terminal 18 percent of the highly-conserved catalytic beta subunit will be examined in a system of chimeric subunits containing regions of the beta subunits from both E. coli and Bacillus megaterium. These chimeras display a distinctive energy-coupling defect which does not affect ATPase activity, but does affect ATP-driven proton pumping and respiration-dependent ATP synthesis. This defect is caused by one or more of the small number of amino acid differences between E. coli and B. megaterium in the last 75-80 residues of the beta subunit. A comparison of those differences to the X-ray structure of the bovine mitochondrial ATPase has identified residues which might influence energy coupling. The proposed research will mutagenize those residues in the megaterium sequences to their E. coli counterparts to identify the residue or residues responsible for the energy coupling defect. Additional mutagenesis experiments will test hypotheses about how those altered amino acids affect inter- and intra-subunit interactions involved in transmitting energy between the beta subunit and the gamma subunit. The gamma subunit transmits energy between the transmembrane Fo proton channel and the alpha and beta subunits of the ATPase. The goal of this research, therefore, is to elucidate the pathway of energy coupling in the ATPase between the proton channel and the catalytic site.