Kinetic isotope effect analysis of the reaction catalyzed by Trypanosoma congolense trypanothione reductase.
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/K for 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 pK 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 2',3'-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.(ABSTRACT TRUNCATED AT 250 WORDS)
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Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461.
Bradley
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Nadeau
K
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Walsh
C T
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Blanchard
J S
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D000818:Animals; D003903:Deuterium; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D011994:Recombinant Proteins; D012997:Solvents; D014316:Tritium; D014348:Trypanosoma congolense / Q000201:enzymology*
D011994:Recombinant Proteins; D012997:Solvents; D014316:Tritium; D009249:NADP; D003903:Deuterium; D009247:NADH, NADPH Oxidoreductases; C049608:trypanothione reductase