Photoaffinity labeling of the coupling factor 1 from the thermophilic bacterum PS3 by 8‐azido ATP

Abstract

To localize the nucleotide binding sites of the F1ATPase (TF1) from the thermophilic bacterium PS3 we have used 14C‐labeled 8‐azido ATP (8‐N3ATP) as photoaffmity label. 8‐N3ATP is hydrolyzed by the F,ATPase in the absence of ultraviolet light. Irradiation by ultraviolet light of the enzyme in the presence of 8‐N3ATP results in reduction of ATPase activity and in preferential nucleotide specific labeling of the α subunits (0.8–0.9 mol 8‐N3ATP/TF1,α:β = 4:1). Inactivation and labeling do not depend on the presence of Mg2+. Both effects decrease upon addition of various nucleotide di‐ or triphosphates.

Molecular mechanics of protonmotive F0F1 ATPases

“…This behaviour is consistent with observations on the relatively constant ahelix and P-sheet content of Fr [4], and with observations indicating that the configurational changes in Fr involve little internal frictional resistance, but depend on the viscosity of the external aqueous medium [47]. The fact that the phosphorylation of ADP requires relatively complete assemblies of functional cy and p subunits with the y subunit (7)(8)(9)(10)13,14,17,20,21] is consistent with the notion (illustrated in fig.2) that each (Y and each p subunit is involved by its rolling action in co-operative activity with the fl subunits and LY subunits, respectively, on either side of it. It is a noteworthy feature of the version of this mechanism shown in fig.2 that, owing to the similar rolling relationships of the p subunits on the y subunit and on the LY subunits, the diametrically asymmetric features of all three cy subunits would be aligned parallel throughout the catalytic configurational cycle.…”

8-N3-3′-Biotinyl-ATP, a Novel Monofunctional Reagent: Differences in the F1- and V1-ATPases by Means of the ATP Analogue

“…Photoirradiation of F 1 -ATPases with 2-azido-ADP resulted in the exclusive labeling of the ␤ subunit (38,39), thereby discriminating between the catalytic ␤ subunits, where the adenine is in contact with a hydrophobic interface (35), and the noncatalytic ␣ subunits, where binding of adenine involves several hydrogen bonds. This is in contrast to 8-N 3 -ATP or -ADP, which was found to photolabel both the ␣ and ␤ subunits of F 1 (42)(43)(44)(45). More notably, amino acid sequence analysis indicated that the nucleotide analogue 2-N 3 -ATP was covalently bound at the residue ␤-Tyr 341 (46) of the adenine pocket of TF 1 and that the analogue 8-N 3 -ATP leads to derivatization of residue ␤-Tyr 307 ((47); see Fig.…”

Untitled

“…This behaviour is consistent with observations on the relatively constant ahelix and P-sheet content of Fr [4], and with observations indicating that the configurational changes in Fr involve little internal frictional resistance, but depend on the viscosity of the external aqueous medium [47]. The fact that the phosphorylation of ADP requires relatively complete assemblies of functional cy and p subunits with the y subunit (7)(8)(9)(10)13,14,17,20,21] is consistent with the notion (illustrated in fig.2) that each (Y and each p subunit is involved by its rolling action in co-operative activity with the fl subunits and LY subunits, respectively, on either side of it. It is a noteworthy feature of the version of this mechanism shown in fig.2 that, owing to the similar rolling relationships of the p subunits on the y subunit and on the LY subunits, the diametrically asymmetric features of all three cy subunits would be aligned parallel throughout the catalytic configurational cycle.…”