Catalytic and potentiometric characterization of E201D and E201Q mutants of Trypanosoma congolense trypanothione reductase

Renjian Zheng, Narimantas Cenas, John S. Blanchard

Research output: Contribution to journalArticle

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Abstract

Trypanothione reductase is a member of the structurally and functionally well-characterized family of flavoprotein reductases, which catalyze the reduced pyridine nucleotide dependent reduction of their disulfide, peroxide, or metal ion substrates. Trypanothione reductase is found in a wide variety of Trypanosoma species, where the enzyme serves physiologically to protect the organism from oxidative stress and assists in maintaining low intracellular levels of hydrogen peroxide. The redox potential of the flavin and the hydride ion transfer reaction of the pro-S hydrogen of NADPH to N5 of FAD have been proposed to be influenced by the presence of a conserved Lys-Glu (K60-E201) ion pair at the bottom of the nicotinamide binding pocket. We have evaluated this hypothesis by making modest substitutions for both the Lys and Glu residues using site-directed mutagenesis. Replacement of the K60 residue with an arginine led to a poorly expressed, and completely inactive, enzyme. Replacement of the Glu201 residue with either a glutamine (E201Q) or an aspartate (E201D) residue led to expressed enzymes which could be readily purified in >20 mg amounts using protocols developed for the WT enzyme, and which had significant residual trypanothione-reducing activity. These enzymes have now been characterized to determine their redox potentials, catalytic activities, and nucleotide specificities. Relative to the WT enzyme, both E201D and E201Q exhibit ca. 5% of WT trypanothione-reducing activity using NADPH as reductant, but significantly enhanced quinone reductase activity. The oxidase activity of both mutants is enhanced by over 50-fold compared to that of the WT. The redox potential of the WT enzyme has been determined to be -273 mV, while both the E201D and E201Q exhibit more positive redox potentials (-259 and -251 mV, respectively). These data confirm the modulating effect of the K60-E201 ion pair on the redox potential of the flavin, and the reactivity of the enzyme toward alternate reducible substrates.

Original languageEnglish (US)
Pages (from-to)12697-12703
Number of pages7
JournalBiochemistry
Volume34
Issue number39
StatePublished - 1995

Fingerprint

Trypanosoma congolense
Oxidation-Reduction
Enzymes
Ions
NADP
lysylglutamic acid
Oxidoreductases
Nucleotides
NAD(P)H Dehydrogenase (Quinone)
Trypanosoma
Flavoproteins
Mutagenesis
Flavin-Adenine Dinucleotide
Oxidative stress
Niacinamide
trypanothione reductase
Reducing Agents
Peroxides
Substrates
Site-Directed Mutagenesis

ASJC Scopus subject areas

  • Biochemistry

Cite this

Catalytic and potentiometric characterization of E201D and E201Q mutants of Trypanosoma congolense trypanothione reductase. / Zheng, Renjian; Cenas, Narimantas; Blanchard, John S.

In: Biochemistry, Vol. 34, No. 39, 1995, p. 12697-12703.

Research output: Contribution to journalArticle

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abstract = "Trypanothione reductase is a member of the structurally and functionally well-characterized family of flavoprotein reductases, which catalyze the reduced pyridine nucleotide dependent reduction of their disulfide, peroxide, or metal ion substrates. Trypanothione reductase is found in a wide variety of Trypanosoma species, where the enzyme serves physiologically to protect the organism from oxidative stress and assists in maintaining low intracellular levels of hydrogen peroxide. The redox potential of the flavin and the hydride ion transfer reaction of the pro-S hydrogen of NADPH to N5 of FAD have been proposed to be influenced by the presence of a conserved Lys-Glu (K60-E201) ion pair at the bottom of the nicotinamide binding pocket. We have evaluated this hypothesis by making modest substitutions for both the Lys and Glu residues using site-directed mutagenesis. Replacement of the K60 residue with an arginine led to a poorly expressed, and completely inactive, enzyme. Replacement of the Glu201 residue with either a glutamine (E201Q) or an aspartate (E201D) residue led to expressed enzymes which could be readily purified in >20 mg amounts using protocols developed for the WT enzyme, and which had significant residual trypanothione-reducing activity. These enzymes have now been characterized to determine their redox potentials, catalytic activities, and nucleotide specificities. Relative to the WT enzyme, both E201D and E201Q exhibit ca. 5{\%} of WT trypanothione-reducing activity using NADPH as reductant, but significantly enhanced quinone reductase activity. The oxidase activity of both mutants is enhanced by over 50-fold compared to that of the WT. The redox potential of the WT enzyme has been determined to be -273 mV, while both the E201D and E201Q exhibit more positive redox potentials (-259 and -251 mV, respectively). These data confirm the modulating effect of the K60-E201 ion pair on the redox potential of the flavin, and the reactivity of the enzyme toward alternate reducible substrates.",
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