Bovine F1-ATPase covalently inhibited with 4-chloro-7-nitrobenzofurazan: the structure provides further support for a rotary catalytic mechanism.

TitleBovine F1-ATPase covalently inhibited with 4-chloro-7-nitrobenzofurazan: the structure provides further support for a rotary catalytic mechanism.
Publication TypeJournal Article
Year of Publication1998
AuthorsOrriss, GL, Leslie, AG, Braig, K, Walker, JE
JournalStructure
Volume6
Issue7
Pagination831-7
Date Published1998 Jul 15
ISSN0969-2126
Keywords4-Chloro-7-nitrobenzofurazan, Animals, Binding Sites, Cattle, Crystallography, X-Ray, Enzyme Inhibitors, Models, Molecular, Protein Conformation, Proton-Translocating ATPases, Tyrosine
Abstract

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.

DOI10.1016/S0969-2126(98)00085-9
Alternate JournalStructure
Citation Key10.1016/S0969-2126(98)00085-9
PubMed ID9687365