The beta G156C substitution in the F1-ATPase from the thermophilic Bacillus PS3 affects catalytic site cooperativity by destabilizing the closed conformation of the catalytic site.
Fluorescence titrations of the alpha(3)(betaG(156)C/Y(345)W)(3)gamma, alpha(3)(betaE(199)V/Y(345)W)(3)gamma, and alpha(3)(betaY(345)W)(3)gamma subcomplexes of TF(1) with nucleotides show that the betaG(156)C substitution substantially lowers the affinity of catalytic sites for ATP and ADP with or without Mg(2+), whereas the betaE(199)V substitution increases the affinity of catalytic sites for nucleotides. Whereas the alpha(3)(betaG(156)C)(3)gamma and alpha(3)(betaE(199)V)(3)gamma subcomplexes hydrolyze 2 mM ATP at 2% and 0.7%, respectively, of the rate exhibited by the wild-type enzyme, the alpha(3)(betaG(156)C/E(199)V)(3)gamma hydrolyzes 2 mM ATP at 9% the rate exhibited by the wild-type enzyme. The alpha(3)(betaG(156)C)(3)gamma, alpha(3)(betaG(156)C/E(199)V)(3)gamma, and alpha(3)(betaG(156)C/E(199)V/Y(345)W)(3)gamma subcomplexes resist entrapment of inhibitory MgADP in a catalytic site during turnover. Product [(3)H]ADP remains tightly bound to a single catalytic site when the wild-type, betaE(199)V, betaY(345)W, and betaE(199)V/Y(345)W subcomplexes hydrolyze substoichiometric [(3)H]ATP, whereas it is not retained by the betaG(156)C and betaG(156)C/Y(345)W subcomplexes. Less firmly bound, product [(3)H]ADP is retained when the betaG(156)C/E(199)V and betaG(156)C/E(199)V/Y(345)W mutants hydrolyze substoichiometric [(3)H]ATP. The Lineweaver-Burk plot obtained with the betaG(156)C mutant is curved downward in a manner indicating that its catalytic sites act independently during ATP hydrolysis. In contrast, the betaG(156)C/E(199)V and betaG(156)C/E(199)V/Y(345)W mutants hydrolyze ATP with linear Lineweaver-Burk plots, indicating cooperative trisite catalysis. It appears that the betaG(156)C substitution destabilizes the closed conformation of a catalytic site hydrolyzing MgATP in a manner that allows release of products in the absence of catalytic site cooperativity. Insertion of the betaE(199)V substitution into the betaG(156)C mutant restores cooperativity by restricting opening of the catalytic site hydrolyzing MgATP for product release until an open catalytic site binds MgATP.
Bandyopadhyay
Sanjay
S
Department of Chemistry & Biochemistry, University of California at San Diego, La Jolla, California 92093-0601, USA.
Valder
Carolina R
CR
Huynh
Hue G
HG
Ren
Huimiao
H
Allison
William S
WS
D000255:Adenosine Triphosphate / Q000378:metabolism; D019943:Amino Acid Substitution / Q000235:genetics*; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics*; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics*; D003545:Cysteine / Q000235:genetics; D004123:Dimethylamines / Q000737:chemistry; D004795:Enzyme Stability / Q000235:genetics; D018698:Glutamic Acid / Q000235:genetics; D005998:Glycine / Q000235:genetics; D006868:Hydrolysis; D008274:Magnesium / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D019810:Sodium Azide / Q000737:chemistry; D014364:Tryptophan / Q000235:genetics; D014443:Tyrosine / Q000235:genetics; D014633:Valine / Q000235:genetics
D004123:Dimethylamines; D018698:Glutamic Acid; D014443:Tyrosine; C014518:dodecyldimethylamine oxide; D014364:Tryptophan; D000255:Adenosine Triphosphate; D019810:Sodium Azide; D006180:Proton-Translocating ATPases; D014633:Valine; D008274:Magnesium; D003545:Cysteine; D005998:Glycine