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

FEBS Letters

Matorin, Andrey D.¹  Mezentzev, Alexander V.³  Fedorchuk, Vladimir V.¹  Tishkov, Vladimir I.¹  Popov, Vladimir O.³  Dementieva, Larissa A.¹  Rojkova, Alexandra M.¹  Lamzin, Victor S.²  Savitsky, Pavel A.¹ 

[1] Department of Chemical Enzymology, Faculty of Chemistry, M. V. Lomonosov Moscow State University, Leninskie Gori, Moscow 119899, Russian Federation;European Molecular Biology Laboratory (EMBL), c/o DESY, Notkestraβe 85, 22603 Hamburg, Germany;A.N. Bakh Institute of Biochemistry, Russian Academy of Sciences, Leninsky pr. 33, Moscow 117071, Russian Federation

关键词: NAD+-dependent formate dehydrogenase;    Active center;    Site-directed mutagenesis;    Molecular mechanism;    FDH;    NAD+-dependent formate dehydrogenase (EC 1.2.1.2);    K d NAD;    dissociation constant of NAD+ from the binary FDH-NAD+ complex;   

DOI  :  10.1016/0014-5793(96)00641-2

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Gln³¹³ and His³³² residues in the active centre of NAD⁺-dependent formate dehydrogenase (EC 1.2.1.2, FDH) from the bacterium Pseudomonas sp. 101 are conserved in all FDHs and are equivalent to the glutamate-histidine pair in active sites of d-specific 2-hydroxyacid dehydrogenases. Two mutants of formate dehydrogenase from Pseudomonas sp. 101, Gln³¹³Glu and His³³²Phe, have been obtained and characterised. The Gln³¹³Glu 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 Gln³¹³ is essential for the broad pH affinity profile towards substrate. His³³²Phe mutation leads to a complete loss of enzyme activity. The His³³²Phe 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 His³³² 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 Gln³¹³ carboxamide.

【 授权许可】

Unknown