Geometric isomers of covalently labeled mitochondrial F1-adenosinetriphosphatase with different properties.
Two geometric isomers of covalently labeled F1-adenosinetriphosphatase (F1-ATPase) have been prepared by reaction with 7-chloro-4-nitro-2,1,3-benzoxadiazole (NBD-Cl): a directly labeled product denoted by O-beta'-NBD-F1 and an indirectly prepared product denoted by 0-beta'-NBD-F1. The normal isomer O-beta'-NBD-F1 is highly inhibited, and its label can be removed by 20 microM N-acetyl-L-cysteine (AC) at the expected rate with dr/dn approximately equal to -1, where n is the molar ratio of the label to F1 and r is the ratio of the ATPase activity of the labeled enzyme to that of the unlabeled control enzyme. But O-beta"-NBD-F1 is almost fully active, and its label can be removed by 20 microM AC at much slower rates with dr/dn approximately equal to 0. Cleavage of either isomer with pepsin and subsequent amino acid analysis of the isolated radioactive polypeptides show that the label is attached to Tyr-beta 311 in both isomers. At pH 9 the label in O-beta'-NBD-F1 spontaneously transfers from Tyr-beta 311 to the presumably nearby Lys-beta 162 in the dark with a half-time of 1/2 h, but the label in O-beta"-NBD-F1 does not transfer under the same conditions. The existence of geometric isomers of O-NBD-F1 with contrastingly different properties invalidates models for F1 with three equivalent beta subunits but is consistent with the model based on one principal catalytic beta' subunit and two auxiliary beta" subunits. A possible mechanism for promoting the catalytic efficiency of beta' through protein conformation change induced by ATP and/or ADP is suggested.
D009327:4-Chloro-7-nitrobenzofurazan / Q000494:pharmacology*; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D007536:Isomerism; D008929:Mitochondria, Heart / Q000201:enzymology*; D010069:Oxadiazoles / Q000494:pharmacology*; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D000596:Amino Acids; D010069:Oxadiazoles; D006180:Proton-Translocating ATPases; D009327:4-Chloro-7-nitrobenzofurazan