Kinetic and chemical mechanisms of yeast formate dehydrogenase

John S. Blanchard, W. W. Cleland

Research output: Contribution to journalArticle

95 Citations (Scopus)

Abstract

Yeast formate dehydrogenase has an ordered kinetic mechanism with NAD adding before formate. NAD analogues are substrates, but formate is the only molecule oxidized. Anions are competitive inhibitors vs. formate and bind only to E-NAD. Linear triatomic anions are the best inhibitors, with azide (Ki = 7 nM) showing tight binding inhibition behavior and appearing to be a transition-state analogue. pH profiles of the kinetic parameters show that a group in E-NAD with a pK of 8.3 must be protonated for binding of azide and formate. Since the pK is elevated to 9.8 upon formate binding, it is probably a cationic acid involved in substrate binding. A group with pK = 6.4 and no temperature dependence must be ionized for binding of azide and formate, and another group with pK = 5.9 must be ionized for catalysis, but the roles of these groups in the mechanism are not clear. A 13C isotope effect of 1.043 with either unlabeled or deuterated formate shows that formate has a very low commitment to catalysis, and all isotope effects observed are intrinsic ones on the chemical reaction. The deuterium isotope effect on V/Kformate varies with the nature of the nucleotide, presumably as the result of changes in transition-state structure. The value of 2.8 with NAD probably results from a late transition state, while the values of 4.4 for thio-NAD, 6.9 for acetylpyridine-NAD, and 3.8 for pyridinecarboxaldehyde-NAD represent progressively earlier transition states. By contrast, the 13C isotope effect drops slightly to 1.036 with acetylpyridine-NAD. These data are explained if both a higher redox potential (that is, more electrophilic C-4) for the nucleotide and an increased distance between formate and nucleotide in the transition state (indicated by a lower Vmax and increased activation energy for the reaction) cause the transition state to become earlier.

Original languageEnglish (US)
Pages (from-to)3543-3550
Number of pages8
JournalBiochemistry
Volume19
Issue number15
StatePublished - 1980
Externally publishedYes

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formic acid
Formate Dehydrogenases
Yeast
NAD
Yeasts
Kinetics
Isotopes
Azides
Nucleotides
Catalysis
Anions
Deuterium

ASJC Scopus subject areas

  • Biochemistry

Cite this

Kinetic and chemical mechanisms of yeast formate dehydrogenase. / Blanchard, John S.; Cleland, W. W.

In: Biochemistry, Vol. 19, No. 15, 1980, p. 3543-3550.

Research output: Contribution to journalArticle

Blanchard, JS & Cleland, WW 1980, 'Kinetic and chemical mechanisms of yeast formate dehydrogenase', Biochemistry, vol. 19, no. 15, pp. 3543-3550.
Blanchard, John S. ; Cleland, W. W. / Kinetic and chemical mechanisms of yeast formate dehydrogenase. In: Biochemistry. 1980 ; Vol. 19, No. 15. pp. 3543-3550.
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