The N-termini of the alpha and beta subunits at the top of F1 stabilize the energy-transfer function in the mitochondrial F1Fo ATP synthase.
Limited trypsin digestion of isolated F1 removed 15 and 7 amino acids from the N-termini of the alpha and beta subunits respectively and left other subunits untouched as shown by electrophoresis, immunoblotting and protein sequencing. The cooperativity for ATP hydrolysis by soluble F1 was impaired by trypsin digestion. The Km2 obtained from Eadie-Hofstee plots apparently decreased in trypsin-digested F1 but the affinity for adenosine 5'-[beta,gamma-imido]triphosphate (AdoPP[NH]P) and GTP hydrolysis was not influenced. The inhibition of ATP hydrolysis by ADP was attenuated by trypsin digestion. Trypsin digestion of F1 did not affect its capacity to bind to Fo nor did it alter the sensitivity of ATP hydrolysis in the F1Fo reconstituted system to oligomycin and N,N'-dicyclohexylcarbodiimide. The cleavage of the alpha and beta subunits did, on the other hand impair: (a) the ATP-driven proton pumping in the reconstituted F1Fo complex: (b) the inhibition by F1 of passive proton conduction in Fo; (c) the inhibition of passive proton conduction in Fo by AdoPP[NH]P binding to F1. These results show that the limited cleavage of the N-termini of the alpha and beta subunits, located on the top of F1, results in decoupling of catalysis from proton transport. The possible relationship of these observations with the binding change rotatory model of the F1Fo ATP synthase is discussed.
Xu
T
T
Institute of Medical Biochemistry and Chemistry, University of Bari, Italy.
Candita
C
C
Amoruso
G
G
Papa
S
S
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000266:Adenylyl Imidodiphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D002417:Cattle; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology*; D008969:Molecular Sequence Data; D009840:Oligomycins / Q000494:pharmacology; D010446:Peptide Fragments / Q000737:chemistry; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000502:physiology*; D011522:Protons; D017421:Sequence Analysis; D014357:Trypsin / Q000378:metabolism; D014508:Urea / Q000494:pharmacology
D005456:Fluorescent Dyes; D009840:Oligomycins; D010446:Peptide Fragments; D011522:Protons; D000266:Adenylyl Imidodiphosphate; D004024:Dicyclohexylcarbodiimide; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D014508:Urea; D014357:Trypsin; D006180:Proton-Translocating ATPases