Reactivity of old yellow enzyme with α-NADPH and other pyridine nucleotide derivatives

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Abstract

The reaction of Old Yellow enzyme (OYE) with pyridine nucleotides has been examined using steady state kinetics, rapid reaction kinetics, and equilibrium binding. α-NADPH, β-NADPH, and the acid breakdown products of NADPH all bind to oxidized OYE with dissociation constants below 1 μM. These complexes produce characteristic red shifts in the absorption spectrum of OYE. A similar red shift which occurs after multiple turnovers of OYE with NADPH has been found to be due to an impurity in the NADPH preparation, possibly an acid breakdown product. Anions such as chloride, acetate, azide, and phenolates compete with the pyridine nucleotides for binding to a common site in oxidized OYE. Anaerobic reduction of OYE by NADPH proceeds via two intermediates to establish a readily reversible equilibrium. In contrast to most other NADPH-dependent enzymes, both α- and β-NADPH are capable of reducing OYE, and α-NADPH is more effective. Using β-[4(R)-2H]NADPH, a primary deuterium isotope effect was observed in the reduction reaction. Results from rapid reaction and steady state studies showed that reduction of OYE was rate limiting in turnover. Consistent with this, the turnover number with αNADPH was significantly higher than that with β-NADPH.

Original languageEnglish (US)
Pages (from-to)1215-1222
Number of pages8
JournalJournal of Biological Chemistry
Volume261
Issue number3
StatePublished - Jan 1 1986

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NADPH Dehydrogenase
NADP
Nucleotides
Derivatives
pyridine
Acids
Azides
Deuterium
Reaction kinetics
Isotopes
Anions
Chlorides
Absorption spectra
Acetates

ASJC Scopus subject areas

  • Biochemistry

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Reactivity of old yellow enzyme with α-NADPH and other pyridine nucleotide derivatives. / Massey, V.; Schopfer, Lawrence M.

In: Journal of Biological Chemistry, Vol. 261, No. 3, 01.01.1986, p. 1215-1222.

Research output: Contribution to journalArticle

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abstract = "The reaction of Old Yellow enzyme (OYE) with pyridine nucleotides has been examined using steady state kinetics, rapid reaction kinetics, and equilibrium binding. α-NADPH, β-NADPH, and the acid breakdown products of NADPH all bind to oxidized OYE with dissociation constants below 1 μM. These complexes produce characteristic red shifts in the absorption spectrum of OYE. A similar red shift which occurs after multiple turnovers of OYE with NADPH has been found to be due to an impurity in the NADPH preparation, possibly an acid breakdown product. Anions such as chloride, acetate, azide, and phenolates compete with the pyridine nucleotides for binding to a common site in oxidized OYE. Anaerobic reduction of OYE by NADPH proceeds via two intermediates to establish a readily reversible equilibrium. In contrast to most other NADPH-dependent enzymes, both α- and β-NADPH are capable of reducing OYE, and α-NADPH is more effective. Using β-[4(R)-2H]NADPH, a primary deuterium isotope effect was observed in the reduction reaction. Results from rapid reaction and steady state studies showed that reduction of OYE was rate limiting in turnover. Consistent with this, the turnover number with αNADPH was significantly higher than that with β-NADPH.",
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N2 - The reaction of Old Yellow enzyme (OYE) with pyridine nucleotides has been examined using steady state kinetics, rapid reaction kinetics, and equilibrium binding. α-NADPH, β-NADPH, and the acid breakdown products of NADPH all bind to oxidized OYE with dissociation constants below 1 μM. These complexes produce characteristic red shifts in the absorption spectrum of OYE. A similar red shift which occurs after multiple turnovers of OYE with NADPH has been found to be due to an impurity in the NADPH preparation, possibly an acid breakdown product. Anions such as chloride, acetate, azide, and phenolates compete with the pyridine nucleotides for binding to a common site in oxidized OYE. Anaerobic reduction of OYE by NADPH proceeds via two intermediates to establish a readily reversible equilibrium. In contrast to most other NADPH-dependent enzymes, both α- and β-NADPH are capable of reducing OYE, and α-NADPH is more effective. Using β-[4(R)-2H]NADPH, a primary deuterium isotope effect was observed in the reduction reaction. Results from rapid reaction and steady state studies showed that reduction of OYE was rate limiting in turnover. Consistent with this, the turnover number with αNADPH was significantly higher than that with β-NADPH.

AB - The reaction of Old Yellow enzyme (OYE) with pyridine nucleotides has been examined using steady state kinetics, rapid reaction kinetics, and equilibrium binding. α-NADPH, β-NADPH, and the acid breakdown products of NADPH all bind to oxidized OYE with dissociation constants below 1 μM. These complexes produce characteristic red shifts in the absorption spectrum of OYE. A similar red shift which occurs after multiple turnovers of OYE with NADPH has been found to be due to an impurity in the NADPH preparation, possibly an acid breakdown product. Anions such as chloride, acetate, azide, and phenolates compete with the pyridine nucleotides for binding to a common site in oxidized OYE. Anaerobic reduction of OYE by NADPH proceeds via two intermediates to establish a readily reversible equilibrium. In contrast to most other NADPH-dependent enzymes, both α- and β-NADPH are capable of reducing OYE, and α-NADPH is more effective. Using β-[4(R)-2H]NADPH, a primary deuterium isotope effect was observed in the reduction reaction. Results from rapid reaction and steady state studies showed that reduction of OYE was rate limiting in turnover. Consistent with this, the turnover number with αNADPH was significantly higher than that with β-NADPH.

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