The lipoamide dehydrogenase from Mycobacterium tuberculosis permits the direct observation of flavin intermediates in catalysis

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

Research output: Contribution to journalArticle

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Abstract

Lipoamide dehydrogenase catalyses the 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 a tightly, but noncovalently, bound FAD and a redox-active disulfide, which cycle between the oxidized and reduced forms during catalysis. The mechanism of reduction of the Mycobacterium tuberculosis lipoamide dehydrogenase by NADH and [4S-2H]-NADH was studied anaerobically at 4 °C and pH 7.5 by stopped-flow spectrophotometry. Three phases of enzyme reduction were observed. The first phase, characterized by a decrease in absorbance at 400-500 nm and an increase in absorbance at 550-700 nm, was fast (kfor = 1260 s-1, krev = 590 s-1) and represents the formation of FADH2·NAD+, an intermediate that has never been observed before in any wild-type lipoamide dehydrogenase. A primary deuterium kinetic isotope effect [D(kfor + krev) ∼ 4.2] was observed on this phase. The second phase, characterized by regain of the absorbance at 400-500 nm, loss of the 550-700 nm absorbance, and gain of 500-550 nm absorbance, was slower (kobs = 200 s-1). This phase represents the intramolecular transfer of electrons from FADH2 to the redox-active disulfide to generate the anaerobically stable two-electron reduced enzyme, EH2. The third phase, characterized by a decrease in absorbance at 400-550 nm, represents the formation of the four-electron reduced form of the enzyme, EH4. The observed rate constant for this phase showed a decreasing NADH concentration dependence, and results from the slow (kfor = 57 s-1 krev = 128 s-1) isomerization of EH2 or slow release of NAD+ before rapid NADH binding and reaction to form EH4. The mechanism of oxidation of EH2 by NAD+ was also investigated under the same conditions. The 530 nm charge-transfer absorbance of EH2 shifted to 600 nm upon NAD+ binding in the dead time of mixing of the stopped-flow instrument and represents formation of the EH2·NAD+ complex. This was followed by two phases. The first phase (kobs = 750 s-1), characterized by a small decrease in absorbance at 435 and 458 nm, probably represents limited accumulation of FADH2·NAD+. The second phase was characterized by an increase in absorbance at 435 and 458 nm and a decrease in absorbance at 530 and 670 nm. The observed rate constant that describes this phase of ∼115 s-1 probably represents the overall rate of formation of Eox and NADH from EH2 and NAD+, and is largely determined by the slower rates of the coupled sequence of reactions preceding flavin oxidation.

Original languageEnglish (US)
Pages (from-to)14580-14590
Number of pages11
JournalBiochemistry
Volume41
Issue number49
DOIs
StatePublished - Dec 10 2002

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Dihydrolipoamide Dehydrogenase
Catalysis
Mycobacterium tuberculosis
NAD
Observation
Electrons
Disulfides
Oxidation
Oxidation-Reduction
Rate constants
Oxidoreductases
Enzymes
4,6-dinitro-o-cresol
Multienzyme Complexes
Acyltransferases
Regain
Flavin-Adenine Dinucleotide
Deuterium
Spectrophotometry
Isomerization

ASJC Scopus subject areas

  • Biochemistry

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The lipoamide dehydrogenase from Mycobacterium tuberculosis permits the direct observation of flavin intermediates in catalysis. / Argyrou, Argyrides; Blanchard, John S.; Palfey, Bruce A.

In: Biochemistry, Vol. 41, No. 49, 10.12.2002, p. 14580-14590.

Research output: Contribution to journalArticle

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abstract = "Lipoamide dehydrogenase catalyses the 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 a tightly, but noncovalently, bound FAD and a redox-active disulfide, which cycle between the oxidized and reduced forms during catalysis. The mechanism of reduction of the Mycobacterium tuberculosis lipoamide dehydrogenase by NADH and [4S-2H]-NADH was studied anaerobically at 4 °C and pH 7.5 by stopped-flow spectrophotometry. Three phases of enzyme reduction were observed. The first phase, characterized by a decrease in absorbance at 400-500 nm and an increase in absorbance at 550-700 nm, was fast (kfor = 1260 s-1, krev = 590 s-1) and represents the formation of FADH2·NAD+, an intermediate that has never been observed before in any wild-type lipoamide dehydrogenase. A primary deuterium kinetic isotope effect [D(kfor + krev) ∼ 4.2] was observed on this phase. The second phase, characterized by regain of the absorbance at 400-500 nm, loss of the 550-700 nm absorbance, and gain of 500-550 nm absorbance, was slower (kobs = 200 s-1). This phase represents the intramolecular transfer of electrons from FADH2 to the redox-active disulfide to generate the anaerobically stable two-electron reduced enzyme, EH2. The third phase, characterized by a decrease in absorbance at 400-550 nm, represents the formation of the four-electron reduced form of the enzyme, EH4. The observed rate constant for this phase showed a decreasing NADH concentration dependence, and results from the slow (kfor = 57 s-1 krev = 128 s-1) isomerization of EH2 or slow release of NAD+ before rapid NADH binding and reaction to form EH4. The mechanism of oxidation of EH2 by NAD+ was also investigated under the same conditions. The 530 nm charge-transfer absorbance of EH2 shifted to 600 nm upon NAD+ binding in the dead time of mixing of the stopped-flow instrument and represents formation of the EH2·NAD+ complex. This was followed by two phases. The first phase (kobs = 750 s-1), characterized by a small decrease in absorbance at 435 and 458 nm, probably represents limited accumulation of FADH2·NAD+. The second phase was characterized by an increase in absorbance at 435 and 458 nm and a decrease in absorbance at 530 and 670 nm. The observed rate constant that describes this phase of ∼115 s-1 probably represents the overall rate of formation of Eox and NADH from EH2 and NAD+, and is largely determined by the slower rates of the coupled sequence of reactions preceding flavin oxidation.",
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N2 - Lipoamide dehydrogenase catalyses the 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 a tightly, but noncovalently, bound FAD and a redox-active disulfide, which cycle between the oxidized and reduced forms during catalysis. The mechanism of reduction of the Mycobacterium tuberculosis lipoamide dehydrogenase by NADH and [4S-2H]-NADH was studied anaerobically at 4 °C and pH 7.5 by stopped-flow spectrophotometry. Three phases of enzyme reduction were observed. The first phase, characterized by a decrease in absorbance at 400-500 nm and an increase in absorbance at 550-700 nm, was fast (kfor = 1260 s-1, krev = 590 s-1) and represents the formation of FADH2·NAD+, an intermediate that has never been observed before in any wild-type lipoamide dehydrogenase. A primary deuterium kinetic isotope effect [D(kfor + krev) ∼ 4.2] was observed on this phase. The second phase, characterized by regain of the absorbance at 400-500 nm, loss of the 550-700 nm absorbance, and gain of 500-550 nm absorbance, was slower (kobs = 200 s-1). This phase represents the intramolecular transfer of electrons from FADH2 to the redox-active disulfide to generate the anaerobically stable two-electron reduced enzyme, EH2. The third phase, characterized by a decrease in absorbance at 400-550 nm, represents the formation of the four-electron reduced form of the enzyme, EH4. The observed rate constant for this phase showed a decreasing NADH concentration dependence, and results from the slow (kfor = 57 s-1 krev = 128 s-1) isomerization of EH2 or slow release of NAD+ before rapid NADH binding and reaction to form EH4. The mechanism of oxidation of EH2 by NAD+ was also investigated under the same conditions. The 530 nm charge-transfer absorbance of EH2 shifted to 600 nm upon NAD+ binding in the dead time of mixing of the stopped-flow instrument and represents formation of the EH2·NAD+ complex. This was followed by two phases. The first phase (kobs = 750 s-1), characterized by a small decrease in absorbance at 435 and 458 nm, probably represents limited accumulation of FADH2·NAD+. The second phase was characterized by an increase in absorbance at 435 and 458 nm and a decrease in absorbance at 530 and 670 nm. The observed rate constant that describes this phase of ∼115 s-1 probably represents the overall rate of formation of Eox and NADH from EH2 and NAD+, and is largely determined by the slower rates of the coupled sequence of reactions preceding flavin oxidation.

AB - Lipoamide dehydrogenase catalyses the 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 a tightly, but noncovalently, bound FAD and a redox-active disulfide, which cycle between the oxidized and reduced forms during catalysis. The mechanism of reduction of the Mycobacterium tuberculosis lipoamide dehydrogenase by NADH and [4S-2H]-NADH was studied anaerobically at 4 °C and pH 7.5 by stopped-flow spectrophotometry. Three phases of enzyme reduction were observed. The first phase, characterized by a decrease in absorbance at 400-500 nm and an increase in absorbance at 550-700 nm, was fast (kfor = 1260 s-1, krev = 590 s-1) and represents the formation of FADH2·NAD+, an intermediate that has never been observed before in any wild-type lipoamide dehydrogenase. A primary deuterium kinetic isotope effect [D(kfor + krev) ∼ 4.2] was observed on this phase. The second phase, characterized by regain of the absorbance at 400-500 nm, loss of the 550-700 nm absorbance, and gain of 500-550 nm absorbance, was slower (kobs = 200 s-1). This phase represents the intramolecular transfer of electrons from FADH2 to the redox-active disulfide to generate the anaerobically stable two-electron reduced enzyme, EH2. The third phase, characterized by a decrease in absorbance at 400-550 nm, represents the formation of the four-electron reduced form of the enzyme, EH4. The observed rate constant for this phase showed a decreasing NADH concentration dependence, and results from the slow (kfor = 57 s-1 krev = 128 s-1) isomerization of EH2 or slow release of NAD+ before rapid NADH binding and reaction to form EH4. The mechanism of oxidation of EH2 by NAD+ was also investigated under the same conditions. The 530 nm charge-transfer absorbance of EH2 shifted to 600 nm upon NAD+ binding in the dead time of mixing of the stopped-flow instrument and represents formation of the EH2·NAD+ complex. This was followed by two phases. The first phase (kobs = 750 s-1), characterized by a small decrease in absorbance at 435 and 458 nm, probably represents limited accumulation of FADH2·NAD+. The second phase was characterized by an increase in absorbance at 435 and 458 nm and a decrease in absorbance at 530 and 670 nm. The observed rate constant that describes this phase of ∼115 s-1 probably represents the overall rate of formation of Eox and NADH from EH2 and NAD+, and is largely determined by the slower rates of the coupled sequence of reactions preceding flavin oxidation.

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