The binding mechanism of the yeast F1-ATPase inhibitory peptide: role of catalytic intermediates and enzyme turnover.
The mechanism of inhibition of yeast mitochondrial F(1)-ATPase by its natural regulatory peptide, IF1, was investigated by correlating the rate of inhibition by IF1 with the nucleotide occupancy of the catalytic sites. Nucleotide occupancy of the catalytic sites was probed by fluorescence quenching of a tryptophan, which was engineered in the catalytic site (beta-Y345W). Fluorescence quenching of a beta-Trp(345) indicates that the binding of MgADP to F(1) can be described as 3 binding sites with dissociation constants of K(d)(1) = 10 +/- 2 nm, K(d2) = 0.22 +/- 0.03 microm, and K(d3) = 16.3 +/- 0.2 microm. In addition, the ATPase activity of the beta-Trp(345) enzyme followed simple Michaelis-Menten kinetics with a corresponding K(m) of 55 microm. Values for the K(d) for MgATP were estimated and indicate that the K(m) (55 microm) for ATP hydrolysis corresponds to filling the third catalytic site on F(1). IF1 binds very slowly to F(1)-ATPase depleted of nucleotides and under unisite conditions. The rate of inhibition by IF1 increased with increasing concentration of MgATP to about 50 mum, but decreased thereafter. The rate of inhibition was half-maximal at 5 microm MgATP, which is 10-fold lower than the K(m) for ATPase. The variations of the rate of IF1 binding are related to changes in the conformation of the IF1 binding site during the catalytic reaction cycle of ATP hydrolysis. A model is proposed that suggests that IF1 binds rapidly, but loosely to F(1) with two or three catalytic sites filled, and is then locked in the enzyme during catalytic hydrolysis of ATP.
Corvest
Vincent
V
Service de Bioénergétique, Département de Biologie Joliot-Curie and CNRS-URA 2096, CEA Saclay, F 91191 Gif-sur-Yvette, France.
Sigalat
Claude
C
Venard
Renée
R
Falson
Pierre
P
Mueller
David M
DM
Haraux
Francis
F
D000244:Adenosine Diphosphate / Q000737:chemistry; D000251:Adenosine Triphosphatases / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D001665:Binding Sites; D001671:Biochemistry / Q000379:methods; D002384:Catalysis; D020134:Catalytic Domain; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008954:Models, Biological; D008956:Models, Chemical; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry; D000097795:ATPase Inhibitory Protein
D000244:Adenosine Diphosphate; D000251:Adenosine Triphosphatases; D000255:Adenosine Triphosphate; D011506:Proteins; D014364:Tryptophan; D000097795:ATPase Inhibitory Protein