Kinetic Isotope Effect Analysis of the Reaction Catalyzed by Trypanosoma congolense Trypanothione Reductase

Betty Leichus, John S. Blanchard, Mark Bradley, Karl Nadeau, Christopher Walsh

Research output: Contribution to journalArticlepeer-review

22 Scopus citations


African trypanosomes are devoid of glutathione reductase activity, and instead contain a unique flavoprotein variant, trypanothione reductase, which acts on a cyclic derivative of glutathione, trypanothione. The high degree of sequence similarity between trypanothione reductase and glutathione reductase, as well as the obvious similarity in the reactions catalyzed, led us to investigate the pH dependence of the kinetic parameters, and the isotopic behavior of trypanothione reductase. The pH dependence of the kinetic parameters V, V/K for NADH, and V/Kior oxidized trypanothione has been determined for trypanothione reductase from Trypanosoma congolense. Both V/K for NADH and the maximum velocity decrease as single groups exhibiting pK values of 8.87 ± 0.09 and 9.45 ± 0.07, respectively, are deprotonated. V/K for oxidized trypanothione, T(S)2, decreases as two groups exhibiting experimentally indistinguishable pAT values of 8.74 ± 0.03 are deprotonated. Variable magnitudes of the primary deuterium kinetic isotope effects on pyridine nucleotide oxidation are observed on V and V/K when different pyridine nucleotide substrates are used, and the magnitude of DV and D(V/K) is independent of the oxidized trypanothione concentration at pH 7.25. Solvent kinetic isotope effects, obtained with 2ʹ/,3ʹ/-cNADPH as the variable substrate, were observed on V only, and plots of V versus mole fraction of D2O(i.e., proton inventory) were linear, and yielded values of 1.3-1.6 for D2OV Solvent kinetic isotope effects obtained with alternate pyridine nucleotides as substrates were also observed on V, and the magnitude of D2OV decreases for each pyridine nucleotide as its maximal velocity relative to that of NADPH oxidation decreases. These data are compared to similar data obtained for glutathione reductase and other flavoprotein reductases.

Original languageEnglish (US)
Pages (from-to)6414-6420
Number of pages7
Issue number28
StatePublished - Feb 1 1992

ASJC Scopus subject areas

  • Biochemistry


Dive into the research topics of 'Kinetic Isotope Effect Analysis of the Reaction Catalyzed by Trypanosoma congolense Trypanothione Reductase'. Together they form a unique fingerprint.

Cite this