EurekaMag
+ Most Popular
Cunninghamia lanceolata plantations in China
Mammalian lairs in paleo ecological studies and palynology
Studies on technological possibilities in utilization of anhydrous milk fat for production of recombined butter-like products
Should right-sided fibroelastomas be operated upon?
Large esophageal lipoma
Apoptosis in the mammalian thymus during normal histogenesis and under various in vitro and in vivo experimental conditions
Poissons characoides nouveaux ou non signales de l'Ilha do Bananal, Bresil
Desensitizing efficacy of Colgate Sensitive Maximum Strength and Fresh Mint Sensodyne dentifrices
Administration of fluid by subcutaneous infusion: revival of a forgotten method
Tundra mosquito control - an impossible dream?
Schizophrenia for primary care providers: how to contribute to the care of a vulnerable patient population
Geochemical pattern analysis; method of describing the Southeastern limestone regional aquifer system
Incidence of low birth weights in a hospital of Mexico City
Tabanidae
Graded management intensity of grassland systems for enhancing floristic diversity
Microbiology and biochemistry of cheese and fermented milk
The ember tetra: a new pygmy characid tetra from the Rio das Mortes, Brazil, Hyphessobrycon amandae sp. n. (Pisces, Characoidei)
Risk factors of contrast-induced nephropathy in patients after coronary artery intervention
Renovation of onsite domestic wastewater in a poorly drained soil
Observations of the propagation velocity and formation mechanism of burst fractures caused by gunshot
Systolic blood pressure in a population of infants in the first year of life: the Brompton study
Haematological studies in rats fed with metanil yellow
Studies on pasteurellosis. I. A new species of Pasteurella encountered in chronic fowl cholera
Dormancy breaking and germination of Acacia salicina Lindl. seeds
therapy of lupus nephritis. a two-year prospective study

The a3(bY341W)3c subcomplex of the F1-ATPase from the thermophilic Bacillus PS3 fails to dissociate ADP when MgATP is hydrolyzed at a single catalytic site and attains maximal velocity when three catalytic sites are saturated with MgATP


The a3(bY341W)3c subcomplex of the F1-ATPase from the thermophilic Bacillus PS3 fails to dissociate ADP when MgATP is hydrolyzed at a single catalytic site and attains maximal velocity when three catalytic sites are saturated with MgATP



Biochemistry (American Chemical Society) 37(47): 757-64



The hydrolytic properties of the a3b3c and mutant a3(bY341W)3c subcomplexes of the TF1-ATPase have been compared. ATPase activity of the mutant is less sensitive to turnover-dependent inhibition by azide, less suppressed by increasing concentrations of Mg2+ during assay, and less stimulated by lauryl dimethylamine oxide (LDAO). Therefore, it has much lower propensity than wild-type to entrap inhibitory MgADP in a catalytic site during turnover. The fluorescence of the introduced tryptophans in the a3(bY341W)3c subcomplex is completely quenched when catalytic sites are saturated with ATP or ADP with or without Mg2+ present. As reported for the bY331W mutant of Escherichia coli F1 (Weber, J., Wilke-Mounts, S., Lee, R. S.-F., Grell, E., Senior, A. E. (1993) J. Biol. Chem. 268, 20126-20133), this provides a direct probe of nucleotide binding to catalytic sites. Addition of stoichiometric MgATP to the mutant subcomplex quenched one-third the tryptophan fluorescence which did not recover after 60 min. This was caused by entrapment of MgADP in a single catalytic site. Titration of catalytic sites of the a3(bY341W)3c subcomplex with MgADP or MgATP revealed Kd's of < 50 nM, about 0.25 mM and about 35 mM. Titrations were not affected by azide, whereas LDAO lowered the affinities of catalytic sites 2 and 3 for MgADP by 5-fold and 2-fold, respectively. During titration with MgATP, LDAO slightly lowered affinity at ATP concentrations below 30 mM and had no effect at ATP concentrations above 30 mM. Maximal velocity was attained when the third catalytic site was titrated with MgATP in the presence or absence of LDAO. The same Kd's for binding MgATP to the (aA396C)3b3(cA22C) mutant were observed before and after inactivating it by cross-linking a to c. This implies that the different affinities of catalytic sites for MgATP do not represent negative cooperativity, but rather represent heterogeneous affinities of catalytic sites dictated by the position of the coiled-coil of the c subunit within the central cavity of the (ab)3 hexamer. Copyright 1998, American Chemical Society.

Please choose payment method:






(PDF emailed within 1 workday: $29.90)

Accession: 009999980

Download citation: RISBibTeXText

Related references

The alpha 3(beta Y341W)3 gamma subcomplex of the F1-ATPase from the thermophilic Bacillus PS3 fails to dissociate ADP when MgATP is hydrolyzed at a single catalytic site and attains maximal velocity when three catalytic sites are saturated with MgATP. Biochemistry 37(47): 16757-16764, 1998

The a3b3c complex of the F1-ATPase from thermophilic Bacillus PS3 containing the aD261N substitution fails to dissociate inhibitory MgADP from a catalytic site when ATP binds to noncatalytic sites. Biochemistry (American Chemical Society) 34: 412-18, 1995

The alpha 3 beta 3 gamma complex of the F1-ATPase from thermophilic Bacillus PS3 containing the alpha D261N substitution fails to dissociate inhibitory MgADP from a catalytic site when ATP binds to noncatalytic sites. Biochemistry 34(50): 16412-16418, 1995

Characteristics of protection by MgADP and MgATP of α3β3γ subcomplex of thermophilic Bacillus PS3 βY341W-mutant F1-ATPase from inhibition by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole support a bi-site mechanism of catalysis. Biochemistry. Biokhimiia 76(11): 1253-1261, 2011

Role of phosphate chain mobility of MgATP in completing the 3-phosphoglycerate kinase catalytic site: binding, kinetic, and crystallographic studies with ATP and MgATP. Biochemistry 43(12): 3436-3449, 2004

Substitution of b-Glu201 in the a3b3c subcomplex of the F1-ATPase from the thermophilic Bacillus PS3 increases the affinity of catalytic sites for nucleotides. The Journal of Biological Chemistry 275(14): 057-63, 2000

The alpha3beta3gamma subcomplex of the F1-ATPase from the thermophilic bacillus PS3 with the betaT165S substitution does not entrap inhibitory MgADP in a catalytic site during turnover. Journal of Biological Chemistry 271(46): 28818-28824, 1996

The a3b3c subcomplex of the F1-ATPase from the thermophilic Bacillus PS3 with the bT165S substitution does not entrap inhibitory MgADP in a catalytic site during turnover. The Journal of Biological Chemistry 271: 818-24, 1996

Substitution of betaGlu(201) in the alpha(3)beta(3)gamma subcomplex of the F(1)-ATPase from the thermophilic Bacillus PS3 increases the affinity of catalytic sites for nucleotides. Journal of Biological Chemistry 275(14): 10057-10063, 2000

The fluorescence spectrum of the introduced tryptophans in the a3(bF155W)3c subcomplex of the F1-ATPase from the thermophilic Bacillus PS3 cannot be used to distinguish between the number of nucleoside di- and triphosphates bound to catalytic sites. The Journal of Biological Chemistry 277(11): 40-7, 2002

The fluorescence spectrum of the introduced tryptophans in the alpha 3(beta F155W)3gamma subcomplex of the F1-ATPase from the thermophilic Bacillus PS3 cannot be used to distinguish between the number of nucleoside di- and triphosphates bound to catalytic sites. Journal of Biological Chemistry 277(11): 9540-9547, 2002

The G 156 C Substitution in the F 1 -Atpase from the Thermophilic Bacillus Ps3 Affects Catalytic Site Cooperativity by Destabilizing the Closed Conformation of the Catalytic Site. Biochemistry 41(48): 14421-14429, 2002

The bG156C Substitution in the F1-ATPase from the Thermophilic Bacillus PS3 Affects Catalytic Site Cooperativity by Destabilizing the Closed Conformation of the Catalytic Site. Biochemistry (American Chemical Society) 41(48): 421-9, 2002

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. Biochemistry 41(48): 14421-14429, 2002

MgATP-concentration dependence of protection of yeast vacuolar V-ATPase from inactivation by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole supports a bi-site catalytic mechanism of ATP hydrolysis. Biochemical and Biophysical Research Communications 423(2): 355-359, 2012