Importance of F1-ATPase residue alpha-Arg-376 for catalytic transition state stabilization.
The functional role of essential residue alpha-Arg-376 in the catalytic site of F1-ATPase was studied. The mutants alpha R376C, alpha R376Q, and alpha R376K were constructed, and combined with the mutation beta Y331W, to investigate catalytic site nucleotide-binding parameters, and to assess catalytic transition state formation by measurement of MgADP-fluoroaluminate binding. Each mutation caused large impairment of ATP synthesis and hydrolysis. Despite the apparent proximity of alpha-Arg-376 to bound nucleoside di- and triphosphate in published X-ray structures, the mutations had little effect on MgADP or MgATP binding affinities, particularly at the highest affinity catalytic site, site 1. Both Cys and Gln mutants abolished transition state formation, demonstrating that alpha-Arg-376 is normally involved at this step of catalysis. A model of the F1-ATPase catalytic transition state structure is presented and discussed. The Lys mutant, although severely impaired, supported transition state formation, suggesting that an additional essential role for the alpha-Arg-376 guanidinium group exists, likely in alpha/beta conformational signal transmission required for steady-state catalysis. Parallels between alpha-Arg-376 and GAP/G-protein "arginine finger" residues are evident.
Nadanaciva
S
S
Department of Biochemistry and Biophysics, University of Rochester Medical Center, New York 14642, USA.
Weber
J
J
Wilke-Mounts
S
S
Senior
A E
AE
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000535:Aluminum / Q000378:metabolism; D001120:Arginine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D003545:Cysteine / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005461:Fluorine / Q000378:metabolism; D005973:Glutamine / Q000235:genetics; D008239:Lysine / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000235:genetics; D014443:Tyrosine / Q000235:genetics
D005973:Glutamine; C053629:fluoroaluminum; D005461:Fluorine; D014443:Tyrosine; D000244:Adenosine Diphosphate; D014364:Tryptophan; D000255:Adenosine Triphosphate; D001120:Arginine; D000535:Aluminum; D006180:Proton-Translocating ATPases; D008239:Lysine; D003545:Cysteine