A new kinetic model for the steady-state reactions of the quinoprotein methanol dehydrogenase from Paracoccus denitrificans

Thomas K Harris, Victor L. Davidson

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

The reactions of methanol dehydrogenase from Paracoccus denitrificans with artificial electron acceptors, ammonia, cyanide, and substrates have been characterized by steady-state kinetic analysis. Phenazine ethosulfate, a commonly used electron acceptor for this enzyme, was shown to exhibit pronounced substrate inhibition with a Ki value approximately 20-fold lower than its Km. Wurster's Blue exhibited only relatively mild substrate inhibition and was deemed a more appropriate electron acceptor. Ammonia was an obligatory activator of the enzyme at low concentrations and inhibited a high concentrations. The Ki value for this inhibition correlated closely with the Kd calculated from a titration of perturbations of the absorption spectrum of methanol dehydrogenase which were caused by the addition of ammonia. Cyanide, which suppressed the peculiar endogenous reaction of methanol dehydrogenase, was also both an activator of substrate-dependent activity and a competitive inhibitor with respect to methanol. Kinetic analysis indicated that the latter two activities corresponded to two distinct binding sites for cyanide. The Ka for cyanide activation correlated closely with the concentration required to inhibit 50% of the endogenous reaction, suggesting that a single binding event is responsible for both of these effects. A model is presented to describe the effects of ammonia and cyanide in the reaction cycle of methanol dehydrogenase, and the physiological relevance of the activation and inhibition by these compounds in vitro is discussed.

Original languageEnglish
Pages (from-to)4362-4368
Number of pages7
JournalBiochemistry
Volume32
Issue number16
StatePublished - Dec 1 1993
Externally publishedYes

Fingerprint

Paracoccus denitrificans
Cyanides
Ammonia
Kinetics
Substrates
Electrons
Tetramethylphenylenediamine
Chemical activation
Enzyme Activators
Titration
Methanol
Absorption spectra
Binding Sites
alcohol dehydrogenase (acceptor)
Enzymes

ASJC Scopus subject areas

  • Biochemistry

Cite this

A new kinetic model for the steady-state reactions of the quinoprotein methanol dehydrogenase from Paracoccus denitrificans. / Harris, Thomas K; Davidson, Victor L.

In: Biochemistry, Vol. 32, No. 16, 01.12.1993, p. 4362-4368.

Research output: Contribution to journalArticle

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