Bovine F1-ATPase covalently inhibited with 4-chloro-7-nitrobenzofurazan: the structure provides further support for a rotary catalytic mechanism.
BACKGROUND
F1-ATPase is the globular domain of F1F0-ATP synthase that catalyses the hydrolysis of ATP to ADP and phosphate. The crystal structure of bovine F1-ATPase has been determined previously to 2.8 A resolution. The enzyme comprises five different subunits in the stoichiometry alpha 3 beta 3 gamma delta epsilon; the three catalytic beta subunits alternate with the three alpha subunits around the centrally located single gamma subunit. To understand more about the catalytic mechanisms, F1-ATPase was inhibited by reaction with 4-chloro-7-nitrobenzofurazan (NBD-Cl) and the structure of the inhibited complex (F1-NBD) determined by X-ray crystallography.


RESULTS
In the structure the three beta subunits adopt a different conformation with different nucleotide occupancy. NBD-Cl reacts with the phenolic oxygen of Tyr311 of the beta E subunit, which contains no bound nucleotide. The two other catalytic subunits beta TP and beta DP contain bound adenylyl-imidodiphosphate (AMP-PNP) and ADP, respectively. The binding site of the NBD moiety does not overlap with the regions of beta E that form the nucleotide-binding pocket in subunits beta TP and beta DP nor does it occlude the nucleotide-binding site. Catalysis appears to be inhibited because neither beta TP nor beta DP can accommodate a Tyr311 residue bearing an NBD group.


CONCLUSIONS
The results presented here are consistent with a rotary catalytic mechanism of ATP synthesis and hydrolysis, which requires the sequential and concerted participation of all three catalytic sites. NBD-Cl inhibits the enzyme by preventing the modified subunit from adopting a conformation that is essential for catalysis to proceed.
Orriss
G L
GL
Medical Research Council, Laboratory of Molecular Biology, Cambridge, UK.
Leslie
A G
AG
Braig
K
K
Walker
J E
JE
D009327:4-Chloro-7-nitrobenzofurazan / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D008958:Models, Molecular; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D014443:Tyrosine
D004791:Enzyme Inhibitors; D014443:Tyrosine; D006180:Proton-Translocating ATPases; D009327:4-Chloro-7-nitrobenzofurazan