To define in detail the molecular mechanism of NAD+-dependent formate dehydrogenase, the pH dependences of various kinetic and spectroscopic parameters have been studied: Vmax, Km (NAD+), Km (formate), inhibition constants for structural analogues of substrate (NO3-) and product (CNS-, CNO-, N3-), CD and fluorescence properties. The value of Vmax, rate-limiting hydride transfer, is nearly constant throughout the entire pH range of enzyme stability (6.0Ő11.2) but decreases below 6. The Km values for both substrates remain constant within the pH range 6Ő10. At pH values below 6 (for the coenzyme) and above 10 (for both substrate and coenzyme) the Km values increase. In the acidic range this change is attributed to the ionization of two carboxy groups (pK approx. 5.5Ő6.0) located at the NAD+-binding site of the enzyme active centre. The pH transition in the basic region (pK 10.5ŷ0.2) has a conformational origin and affects the enzyme's affinity for substrates and anion inhibitors. A similar transition has been observed for formate dehydrogenases from yeast Candida boidinii and Hansenula polymorpha. The results complement the conclusions about the catalytic mechanism deduced from the crystal structure of the enzyme.
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January 1997
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Research Article|
January 15 1997
Effect of pH on kinetic parameters of NAD+-dependent formate dehydrogenase
Alexander V. MESENTSEV;
Alexander V. MESENTSEV
*A. N. Bakh Institute of Biochemistry, Russian Academy of Sciences, Leninsky pr. 33, Moscow 117071, Russia
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Victor S. LAMZIN;
Victor S. LAMZIN
†European Molecular Biology Laboratory (EMBL), c/o DESY, Notkestrasse 85, 22603, Hamburg, Federal Republic of Germany, Russia
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Vladimir I. TISHKOV;
Vladimir I. TISHKOV
‡Department of Chemical Enzymology, M.V. Lomonosov State University, Leninskie Gori, Moscow 119899, Russia
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Tatyana B. USTINNIKOVA;
Tatyana B. USTINNIKOVA
*A. N. Bakh Institute of Biochemistry, Russian Academy of Sciences, Leninsky pr. 33, Moscow 117071, Russia
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Vladimir O. POPOV
Vladimir O. POPOV
§
*A. N. Bakh Institute of Biochemistry, Russian Academy of Sciences, Leninsky pr. 33, Moscow 117071, Russia
§To whom correspondence should be addressed.
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Publisher: Portland Press Ltd
Received:
May 08 1996
Revision Received:
August 15 1996
Accepted:
August 29 1996
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1997
1997
Biochem J (1997) 321 (2): 475–480.
Article history
Received:
May 08 1996
Revision Received:
August 15 1996
Accepted:
August 29 1996
Citation
Alexander V. MESENTSEV, Victor S. LAMZIN, Vladimir I. TISHKOV, Tatyana B. USTINNIKOVA, Vladimir O. POPOV; Effect of pH on kinetic parameters of NAD+-dependent formate dehydrogenase. Biochem J 15 January 1997; 321 (2): 475–480. doi: https://doi.org/10.1042/bj3210475
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