F1-ATPase alpha-subunit made up from two fragments (1-395, 396-503) is stabilized by ATP and complexes containing it obey altered kinetics.
Inferred from the crystal structure of mitochondrial F1-ATPase (Abrahams, J.P. et al. (1994) Nature 370, 621-628), the proteinase-sensitive region around Phe-395 of thermophilic F1-ATPase alpha-subunit corresponds to the loop which connects main part of the carboxyl-terminal helical bundle domain with the ATP binding domain. This loop is in contact with the gamma- and adjacent beta-subunits. Two polypeptides corresponding to the sequence 1-395 and 396-503 of the alpha-subunit were expressed in Escherichia coli cells and they were copurified as an apparently functional alpha-subunit (alpha(395/396)) made up of two polypeptides. The isolated alpha(395/396) was stabilized by ATP-Mg, but not by ADP-Mg, although it bound both ATP-Mg and ADP-Mg with similar affinities (Kd, 11 microM and 14 microM, respectively). The alpha(395/396) was reconstitutable into alpha(395/396)3 beta 3 and alpha(395/396)3 beta 3 gamma complexes. Different from the intact the ATP-Mg-induced dissociation into alpha 1 beta 1 heterodimers. ATP hydrolysis by the alpha(395/396)3 beta 3 gamma complex underwent a slow initial phase, whereas the intact alpha 3 beta 3 gamma complex exhibited an accelerated initial phase. Steady-state ATPase activity at various ATP concentrations showed negative cooperativity for the intact alpha 3 beta 3 gamma complex but apparently positive cooperativity for the alpha(395/396)3 beta 3 gamma complex. The ATPase activities at a saturating ATP concentration of the complexes containing the alpha(395/396) were 180% of those containing intact alpha-subunits. These results indicate that a loop around Phe-395 is involved in intersubunit interaction in F1-ATPase.
Miyauchi
M
M
Research Laboratory of Resources Utilization, Tokyo Institute of Technology, Yokohama, Japan.
Tozawa
K
K
Yoshida
M
M
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins
D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases