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Tishkov, Vladimir I.; Matorin, Andrey D.; Rojkova, Alexandra M.; Fedorchuk, Vladimir V.; Savitsky, Pavel A.; Dementieva, Larissa A.; Lamzin, Victor S.; Mezentzev, Alexander V.; Popov, Vladimir O.

Site‐directed mutagenesis of the formate dehydrogenase active centre: role of the His332‐Gln313 pair in enzyme catalysis

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  • Medientyp: E-Artikel
  • Titel: Site‐directed mutagenesis of the formate dehydrogenase active centre: role of the His332‐Gln313 pair in enzyme catalysis
  • Beteiligte: Tishkov, Vladimir I.; Matorin, Andrey D.; Rojkova, Alexandra M.; Fedorchuk, Vladimir V.; Savitsky, Pavel A.; Dementieva, Larissa A.; Lamzin, Victor S.; Mezentzev, Alexander V.; Popov, Vladimir O.
  • Erschienen: Wiley, 1996
  • Erschienen in: FEBS Letters
  • Sprache: Englisch
  • DOI: 10.1016/0014-5793(96)00641-2
  • ISSN: 0014-5793; 1873-3468
  • Schlagwörter: Cell Biology ; Genetics ; Molecular Biology ; Biochemistry ; Structural Biology ; Biophysics
  • Entstehung:
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  • Beschreibung: <jats:p>Gln<jats:sup>313</jats:sup> and His<jats:sup>332</jats:sup> residues in the active centre of NAD<jats:sup>+</jats:sup>‐dependent formate dehydrogenase (EC 1.2.1.2, FDH) from the bacterium <jats:italic>Pseudomonas</jats:italic> sp. 101 are conserved in all FDHs and are equivalent to the glutamate‐histidine pair in active sites of <jats:sc>d</jats:sc>‐specific 2‐hydroxyacid dehydrogenases. Two mutants of formate dehydrogenase from <jats:italic>Pseudomonas</jats:italic> sp. 101, Gln<jats:sup>313</jats:sup>Glu and His<jats:sup>332</jats:sup>Phe, have been obtained and characterised. The Gln<jats:sup>313</jats:sup>Glu mutation shifts the pK of the group controlling formate binding from less than 5.5 in wild‐type enzyme to 7.6 thus indicating that Gln<jats:sup>313</jats:sup> is essential for the broad pH affinity profile towards substrate. His<jats:sup>332</jats:sup>Phe mutation leads to a complete loss of enzyme activity. The His<jats:sup>332</jats:sup>Phe mutant is still able to bind coenzyme but not substrate or analogues. The role of histidine in the active centre of FDH is discussed. The protonation state of His<jats:sup>332</jats:sup> is not critical for catalysis but vital for substrate binding. A partial positive charge on the histidine imidazole, required for substrate binding, is provided via tight H‐bond to the Gln<jats:sup>313</jats:sup> carboxamide.</jats:p>
  • Zugangsstatus: Freier Zugang