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Titolo:
Mg2+ is not catalytically required in the intrinsic and kirromycin-stimulated GTPase action of Thermus thermophilus EF-Tu
Autore:
Rutthard, H; Banerjee, A; Makinen, MW;
Indirizzi:
Univ Chicago, Dept Biochem & Mol Biol, Cummings Life Sci Ctr, Chicago, IL 60637 USA Univ Chicago Chicago IL USA 60637 ngs Life Sci Ctr, Chicago, IL 60637 USA Univ Bayreuth, Biochem Lab, D-95440 Bayreuth, Germany Univ Bayreuth Bayreuth Germany D-95440 em Lab, D-95440 Bayreuth, Germany
Titolo Testata:
JOURNAL OF BIOLOGICAL CHEMISTRY
fascicolo: 22, volume: 276, anno: 2001,
pagine: 18728 - 18733
SICI:
0021-9258(20010601)276:22<18728:MINCRI>2.0.ZU;2-C
Fonte:
ISI
Lingua:
ENG
Soggetto:
ELONGATION-FACTOR-TU; ELECTRON-PARAMAGNETIC-RESONANCE; ESCHERICHIA-COLI; CRYSTAL-STRUCTURE; BACILLUS-STEAROTHERMOPHILUS; ANGSTROM RESOLUTION; NUCLEOTIDE BINDING; EFFECTOR REGION; TS COMPLEX; P21 RAS;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
51
Recensione:
Indirizzi per estratti:
Indirizzo: Makinen, MW Univ Chicago, Dept Biochem & Mol Biol, Cummings Life Sci Ctr, 920 E 58Th St, Chicago, IL 60637 USA Univ Chicago 920 E 58Th St Chicago IL USA 60637 , IL 60637 USA
Citazione:
H. Rutthard et al., "Mg2+ is not catalytically required in the intrinsic and kirromycin-stimulated GTPase action of Thermus thermophilus EF-Tu", J BIOL CHEM, 276(22), 2001, pp. 18728-18733

Abstract

The influence of divalent metal ions on the intrinsic and kirromycin-stimulated GTPase activity in the absence of programmed ribosomes and on nucleotide binding affinity of elongation factor Tu (EF-Tu) from Thermus thermophilus prepared as the nucleotide- and Mg2+-free protein has been investigated. The intrinsic GTPase activity under single turnover conditions varied according to the series: Mn2+ (0.069 min(-1)) > Mg2+ (0.037 min(-1)) similar to no Me2+ (0.034 min(-1)) > VO2+ (0.014 min(-1)). The kirromycin-stimulatedactivity showed a parallel variation. Under multiple turnover conditions (GTP/EF-Tu ratio of 10:1), Mg2+ retarded the rate of hydrolysis in comparison to that in the absence of divalent metal ions, an effect ascribed to kinetics of nucleotide exchange, In the absence of added divalent metal ions, GDP and GTP were bound with equal affinity (R, similar to 10(-7) hr). In thepresence of added divalent metal ions, GDP affinity increased by up to twoorders of magnitude according to the series: no Me2+ < VO2+ < Mn2+ similarto Mg2+ whereas the binding affinity of G;TP increased by one order of magnitude: no Me2+ < Mg2+ < VO2+ < Mn2+ Estimates of equilibrium (dissociation) binding constants for GDP and GTP by EF-Tu on the basis of Scatchard plotanalysis, together with thermodynamic data for hydrolysis of triphosphate nucleotides (Phillips, R, C,, George, P., and Rutman, R, J; (1969) J, Biol,Chem, 244, 3330-3342), showed that divalent metal ions stabilize the EF-Tu. Me2+ GDP complex over the protein-free Me2+ GDP complex in solution, with the effect greatest in the presence of Mg2+ by similar to 10 kJ/mol. These combined results show that Mg2+ is not a catalytically obligatory cofactor in intrinsic and kirromycin-stimulated GTPase action of EF-Tu in the absence of programmed ribosomes, which highlights the differential role of Mg2in EF-Tu function.

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Documento generato il 03/07/20 alle ore 00:58:45