Catalysis of diaphorase reactions by Mycobacterium tuberculosis lipoamide dehydrogenase occurs at the EH4 level

Argyrides Argyrou, Guangxing Sun, Bruce A. Palfey, John S. Blanchard

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

Lipoamide dehydrogenase catalyzes the reversible NAD+-dependent oxidation of the dihydrolipoyl cofactors that are covalently attached to the acyltransferase components of the pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, and glycine reductase multienzyme complexes. It contains two redox centers: a tightly, but noncovalently, bound FAD and an enzymic disulfide, each of which can accommodate two electrons. In the two-electron-reduced enzyme (EH2), the disulfide is reduced while the FAD cofactor is oxidized. In the four-electron-reduced enzyme (EH4), both redox centers are reduced. Lipoamide dehydrogenase can also catalyze the NADH-dependent reduction of alternative electron acceptors such as 2,6-dichlorophenolindophenol, ferricyanide, quinones, and molecular oxygen (O2). To determine the mechanism of these "diaphorase" reactions, we generated the EH2 and EH4 forms of Mycobacterium tuberculosis lipoamide dehydrogenase and rapidly mixed these enzyme forms with D,L-lipoylpentanoate, 2,6-dimethyl-1,4-benzoquinone, and O2, in a stopped-flow spectrophotometer at pH 7.5 and 4 °C. EH2 reduced D,L-lipoylpentanoate ≥100 times faster than EH4 did. Conversely, EH4 reduced 2,6-dimethyl-1,4-benzoquinone and molecular oxygen 90 and 40 times faster than EH2, respectively. Comparison of the rates of reduction of the above substrates by EH2 and EH4 with their corresponding steady-state kinetic parameters for kinetic competence leads to the conclusion that reduction of lipoyl substrates occurs with EH2 while reduction of diaphorase substrates occurs with EH4.

Original languageEnglish (US)
Pages (from-to)2218-2228
Number of pages11
JournalBiochemistry
Volume42
Issue number7
DOIs
StatePublished - Feb 25 2003

Fingerprint

Dihydrolipoamide Dehydrogenase
Catalysis
Mycobacterium tuberculosis
Electrons
Flavin-Adenine Dinucleotide
Molecular oxygen
Disulfides
NAD
Oxidation-Reduction
Oxidoreductases
Substrates
Enzymes
2,6-Dichloroindophenol
Multienzyme Complexes
Oxygen
Acyltransferases
Quinones
Spectrophotometers
Pyruvic Acid
Kinetic parameters

ASJC Scopus subject areas

  • Biochemistry

Cite this

Catalysis of diaphorase reactions by Mycobacterium tuberculosis lipoamide dehydrogenase occurs at the EH4 level. / Argyrou, Argyrides; Sun, Guangxing; Palfey, Bruce A.; Blanchard, John S.

In: Biochemistry, Vol. 42, No. 7, 25.02.2003, p. 2218-2228.

Research output: Contribution to journalArticle

Argyrou, Argyrides ; Sun, Guangxing ; Palfey, Bruce A. ; Blanchard, John S. / Catalysis of diaphorase reactions by Mycobacterium tuberculosis lipoamide dehydrogenase occurs at the EH4 level. In: Biochemistry. 2003 ; Vol. 42, No. 7. pp. 2218-2228.
@article{3f0a675a464f47a8bdfc3f5aabbec7c5,
title = "Catalysis of diaphorase reactions by Mycobacterium tuberculosis lipoamide dehydrogenase occurs at the EH4 level",
abstract = "Lipoamide dehydrogenase catalyzes the reversible NAD+-dependent oxidation of the dihydrolipoyl cofactors that are covalently attached to the acyltransferase components of the pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, and glycine reductase multienzyme complexes. It contains two redox centers: a tightly, but noncovalently, bound FAD and an enzymic disulfide, each of which can accommodate two electrons. In the two-electron-reduced enzyme (EH2), the disulfide is reduced while the FAD cofactor is oxidized. In the four-electron-reduced enzyme (EH4), both redox centers are reduced. Lipoamide dehydrogenase can also catalyze the NADH-dependent reduction of alternative electron acceptors such as 2,6-dichlorophenolindophenol, ferricyanide, quinones, and molecular oxygen (O2). To determine the mechanism of these {"}diaphorase{"} reactions, we generated the EH2 and EH4 forms of Mycobacterium tuberculosis lipoamide dehydrogenase and rapidly mixed these enzyme forms with D,L-lipoylpentanoate, 2,6-dimethyl-1,4-benzoquinone, and O2, in a stopped-flow spectrophotometer at pH 7.5 and 4 °C. EH2 reduced D,L-lipoylpentanoate ≥100 times faster than EH4 did. Conversely, EH4 reduced 2,6-dimethyl-1,4-benzoquinone and molecular oxygen 90 and 40 times faster than EH2, respectively. Comparison of the rates of reduction of the above substrates by EH2 and EH4 with their corresponding steady-state kinetic parameters for kinetic competence leads to the conclusion that reduction of lipoyl substrates occurs with EH2 while reduction of diaphorase substrates occurs with EH4.",
author = "Argyrides Argyrou and Guangxing Sun and Palfey, {Bruce A.} and Blanchard, {John S.}",
year = "2003",
month = "2",
day = "25",
doi = "10.1021/bi020654f",
language = "English (US)",
volume = "42",
pages = "2218--2228",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "7",

}

TY - JOUR

T1 - Catalysis of diaphorase reactions by Mycobacterium tuberculosis lipoamide dehydrogenase occurs at the EH4 level

AU - Argyrou, Argyrides

AU - Sun, Guangxing

AU - Palfey, Bruce A.

AU - Blanchard, John S.

PY - 2003/2/25

Y1 - 2003/2/25

N2 - Lipoamide dehydrogenase catalyzes the reversible NAD+-dependent oxidation of the dihydrolipoyl cofactors that are covalently attached to the acyltransferase components of the pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, and glycine reductase multienzyme complexes. It contains two redox centers: a tightly, but noncovalently, bound FAD and an enzymic disulfide, each of which can accommodate two electrons. In the two-electron-reduced enzyme (EH2), the disulfide is reduced while the FAD cofactor is oxidized. In the four-electron-reduced enzyme (EH4), both redox centers are reduced. Lipoamide dehydrogenase can also catalyze the NADH-dependent reduction of alternative electron acceptors such as 2,6-dichlorophenolindophenol, ferricyanide, quinones, and molecular oxygen (O2). To determine the mechanism of these "diaphorase" reactions, we generated the EH2 and EH4 forms of Mycobacterium tuberculosis lipoamide dehydrogenase and rapidly mixed these enzyme forms with D,L-lipoylpentanoate, 2,6-dimethyl-1,4-benzoquinone, and O2, in a stopped-flow spectrophotometer at pH 7.5 and 4 °C. EH2 reduced D,L-lipoylpentanoate ≥100 times faster than EH4 did. Conversely, EH4 reduced 2,6-dimethyl-1,4-benzoquinone and molecular oxygen 90 and 40 times faster than EH2, respectively. Comparison of the rates of reduction of the above substrates by EH2 and EH4 with their corresponding steady-state kinetic parameters for kinetic competence leads to the conclusion that reduction of lipoyl substrates occurs with EH2 while reduction of diaphorase substrates occurs with EH4.

AB - Lipoamide dehydrogenase catalyzes the reversible NAD+-dependent oxidation of the dihydrolipoyl cofactors that are covalently attached to the acyltransferase components of the pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, and glycine reductase multienzyme complexes. It contains two redox centers: a tightly, but noncovalently, bound FAD and an enzymic disulfide, each of which can accommodate two electrons. In the two-electron-reduced enzyme (EH2), the disulfide is reduced while the FAD cofactor is oxidized. In the four-electron-reduced enzyme (EH4), both redox centers are reduced. Lipoamide dehydrogenase can also catalyze the NADH-dependent reduction of alternative electron acceptors such as 2,6-dichlorophenolindophenol, ferricyanide, quinones, and molecular oxygen (O2). To determine the mechanism of these "diaphorase" reactions, we generated the EH2 and EH4 forms of Mycobacterium tuberculosis lipoamide dehydrogenase and rapidly mixed these enzyme forms with D,L-lipoylpentanoate, 2,6-dimethyl-1,4-benzoquinone, and O2, in a stopped-flow spectrophotometer at pH 7.5 and 4 °C. EH2 reduced D,L-lipoylpentanoate ≥100 times faster than EH4 did. Conversely, EH4 reduced 2,6-dimethyl-1,4-benzoquinone and molecular oxygen 90 and 40 times faster than EH2, respectively. Comparison of the rates of reduction of the above substrates by EH2 and EH4 with their corresponding steady-state kinetic parameters for kinetic competence leads to the conclusion that reduction of lipoyl substrates occurs with EH2 while reduction of diaphorase substrates occurs with EH4.

UR - http://www.scopus.com/inward/record.url?scp=0037465428&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0037465428&partnerID=8YFLogxK

U2 - 10.1021/bi020654f

DO - 10.1021/bi020654f

M3 - Article

C2 - 12590611

AN - SCOPUS:0037465428

VL - 42

SP - 2218

EP - 2228

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 7

ER -