Browsing by Subject "Adenosine Triphosphate"

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    Asparagine 706 and Glutamate 183 at the Catalytic Site of Sarcoplasmic Reticulum Ca 2+ -ATPase Play Critical but Distinct Roles in E 2 States
    (2006) Clausen, Johannes D; McIntosh, David B; Woolley, David G; Anthonisen, Anne Nyholm; Vilsen, Bente; Andersen, Jens Peter
    Mutants with alteration to Asn(706) of the highly conserved (701)TGDGVND(707) motif in domain P of sarcoplasmic reticulum Ca(2+)-ATPase were analyzed for changes in transport cycle kinetics and binding of the inhibitors vanadate, BeF, AlF, and MgF. The fluorides likely mimic the phosphoryl group/P(i) in the respective ground, transition, and product states of phosphoenzyme hydrolysis (Danko, S., Yamasaki, K., Daiho, T., and Suzuki, H. (2004) J. Biol. Chem. 279, 14991-14998). Binding of BeF, AlF, and MgF was also studied for mutant Glu(183) --> Ala, where the glutamate of the (181)TGES(184) motif in domain A is replaced. Mutations of Asn(706) and Glu(183) have in common that they dramatically impede the function of the enzyme in E2 states, but have little effect in E1. Contrary to the Glu(183) mutant, in which E2P slowly accumulates (Clausen, J. D., Vilsen, B., McIntosh, D. B., Einholm, A. P., and Andersen, J. P. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 2776-2781), E2P formation was not detectable with the Asn(706) mutants. Differential sensitivities of the mutants to inhibition by AlF, MgF, and BeF made it possible to distinguish different roles of Asn(706) and Glu(183). Hence, Asn(706) is less important than Glu(183) for gaining the transition state during E2P hydrolysis but plays critical roles in stabilization of E2P ground and E2.P(i) product states and in the major conformational changes associated with the Ca(2)E1P --> E2P and E2 --> Ca(2)E1 transitions, which seem to be facilitated by interaction of Asn(706) with domain A.
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    ATPase and Multidrug Transport Activities of the Overexpressed Yeast ABC Protein Yor1p
    (1998) Decottignies, Anabelle; Grant, Althea M; Nichols, J Wylie; de Wet, Heidi; McIntosh, David B; Goffeau, André
    The Saccharomyces cerevisiae genome encodes 15 full-size ATP binding cassette transporters (ABC), of which PDR5, SNQ2, and YOR1 are known to be regulated by the transcription factors Pdr1p and Pdr3p (pleiotropic drug resistance). We have identified two new ABC transporter-encoding genes, PDR10 and PDR15, which were up-regulated by the PDR1-3 mutation. These genes, as well as four other ABC transporter-encoding genes, were deleted in order to study the properties of Yor1p. The PDR1-3 gain-of-function mutant was then used to overproduce Yor1p up to 10% of the total plasma membrane proteins. Overexpressed Yor1p was photolabeled by [gamma-32P]2', 3'-O-(2,4,6-trinitrophenyl)-8-azido-ATP (K0.5 = 45 microM) and inhibited by ATP (KD = 0.3 mM) in plasma membranes. Solubilization and partial purification on sucrose gradient allowed to detect significant Yor1p ATP hydrolysis activity (approximately 100 nmol of Pi.min-1.mg-1). This activity was phospholipid-dependent and sensitive to low concentrations of vanadate (I50 = 0.3 microM) and oligomycin (I50 = 8.5 microg/ml). In vivo, we observed a correlation between the amount of Yor1p in the plasma membrane and the level of resistance to oligomycin. We also demonstrated that Yor1p drives an energy-dependent, proton uncoupler-insensitive, cellular extrusion of rhodamine B. Furthermore, cells lacking both Yor1p and Pdr5p (but not Snq2p) showed increased accumulation of the fluorescent derivative of 1-myristoyl-2-[6-(NBD)aminocaproyl]phosphatidylethanolamine. Despite their different topologies, both Yor1p and Pdr5p mediated the ATP-dependent translocation of similar drugs and phospholipids across the yeast cell membrane. Both ABC transporters exhibit ATP hydrolysis in vitro, but Pdr5p ATPase activity is about 15 times higher than that of Yor1p, which may indicate mechanistic or regulatory differences between the two enzymes.
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    Importance of Conserved N-domain Residues Thr 441 , Glu 442 , Lys 515 , Arg 560 , and Leu 562 of Sarcoplasmic Reticulum Ca 2+ -ATPase for MgATP Binding and Subsequent Catalytic Steps: PLASTICITY OF THE NUCLEOTIDE-BINDING SITE
    (2003) Clausen, Johannes D; McIntosh, David B; Vilsen, Bente; Woolley, David G; Andersen, Jens Peter
    Nine single mutations were introduced to amino acid residues Thr441, Glu442, Lys515, Arg560, Cys561, and Leu562 located in the nucleotide-binding domain of sarcoplasmic reticulum Ca2+-ATPase, and the functional consequences were studied in a direct nucleotide binding assay, as well as by steady-state and transient kinetic measurements of the overall and partial reactions of the transport cycle. Some partial reaction steps were also examined in mutants with alterations to Phe487, Arg489, and Lys492. The results implicate all these residues, except Cys561, in high affinity nucleotide binding at the substrate site. Mutations Thr441 --> Ala, Glu442 --> Ala, and Leu562 --> Phe were more detrimental to MgATP binding than to ATP binding, thus pointing to a role for these residues in the binding of Mg2+ or to a difference between the interactions with MgATP and ATP. Subsequent catalytic steps were also selectively affected by the mutations, showing the involvement of the nucleotide-binding domain in these reactions. Mutation of Arg560 inhibited phosphoryl transfer but enhanced the E1PCa2 --> E2P conformational transition, whereas mutations Thr441 --> Ala, Glu442 --> Ala, Lys492 --> Leu, and Lys515 --> Ala inhibited the E1PCa2 --> E2P transition. Hydrolysis of the E2P phosphoenzyme intermediate was enhanced in Glu442 --> Ala, Lys492 --> Leu, Lys515 --> Ala, and Arg560 --> Glu. None of the mutations affected the low affinity activation by nucleotide of the phosphoenzyme-processing steps, indicating that modulatory nucleotide interacts differently from substrate nucleotide. Mutation Glu442 --> Ala greatly enhanced reaction of Lys515 with fluorescein isothiocyanate, indicating that the two residues form a salt link in the native protein.
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    Roles of Conserved P Domain Residues and Mg 2+ in ATP Binding in the Ground and Ca 2+-activated States of Sarcoplasmic Reticulum Ca 2+-ATPase
    (2004) McIntosh, David B; Clausen, Johannes D; Woolley, David G; MacLennan, David H; Vilsen, Bente; Andersen, Jens Peter
    Residues in conserved motifs (625)TGD, (676)FARXXPXXK, and (701)TGDGVND in domain P of sarcoplasmic reticulum Ca(2+)-ATPase, as well as in motifs (601)DPPR and (359)NQR(/K)MSV in the hinge segments connecting domains N and P, were examined by mutagenesis to assess their roles in nucleotide and Mg(2+) binding and stabilization of the Ca(2+)-activated transition state for phosphoryl transfer. In the absence of Mg(2+), mutations removing the charges of domain P residues Asp(627), Lys(684), Asp(703), and Asp(707) increased the affinity for ATP and 2',3'-O-(2,4,6-trinitrophenyl)-8-azidoadenosine 5'-triphosphate. These mutations, as well as Gly(626)--> Ala, were inhibitory for ATP binding in the presence of Mg(2+) and for tight binding of the beta,gamma-bidentate chromium(III) complex of ATP. The hinge mutations had pronounced, but variable, effects on ATP binding only in the presence of Mg(2+). The data demonstrate an unfavorable electrostatic environment for binding of negatively charged nucleotide in domain P and show that Mg(2+) is required to anchor the phosphoryl group of ATP at the phosphorylation site. Mutants Gly(626) --> Ala, Lys(684) --> Met, Asp(703) --> Ala/Ser/Cys, and mutants with alteration to Asp(707) exhibited very slow or negligible phosphorylation, making it possible to measure ATP binding in the pseudo-transition state attained in the presence of both Mg(2+) and Ca(2+). Under these conditions, ATP binding was almost completely blocked in Gly(626) --> Ala and occurred with 12- and 7-fold reduced affinities in Asp(703) --> Ala and Asp(707) --> Cys, respectively, relative to the situation in the presence of Mg(2+) without Ca(2+), whereas in Lys(684) --> Met and Asp(707) --> Ser/Asn the affinity was enhanced 14- and 3-5-fold, respectively. Hence, Gly(626) and Asp(703) seem particularly critical for mediating entry into the transition state for phosphoryl transfer upon Ca(2+) binding at the transport sites.