title: Effect of pH on kinetic parameters of NAD+-dependent formate dehydrogenase.
abstract: 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 (6.0-11.2) but decreases below 6. The K(m) values for both substrates remain constant within the pH range 6-10. 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. 5.5-6.0) located at the NAD+-binding site of the enzyme active centre. The pH transition in the basic region (pK 10.5 +/- 0.2) 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.
authors.affiliation: A. N. Bakh Institute of Biochemistry, Russian Academy of Sciences, Moscow.
authors.forename: A V
authors.initials: AV
authors.lastname: Mesentsev
authors.forename: V S
authors.initials: VS
authors.lastname: Lamzin
authors.forename: V I
authors.initials: VI
authors.lastname: Tishkov
authors.forename: T B
authors.initials: TB
authors.lastname: Ustinnikova
authors.forename: V O
authors.initials: VO
authors.lastname: Popov
mesh_terms: D001665:Binding Sites; D002384:Catalysis; D005560:Formate Dehydrogenases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D010843:Pichia / Q000201:enzymology; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D009243:NAD; D005560:Formate Dehydrogenases