Mycobacterium tuberculosis mycothione reductase: pH dependence of the kinetic parameters and kinetic isotope effects.
The recent identification of the enzyme in Mycobacterium tuberculosis that catalyzes the NADPH-dependent reduction of the unique low molecular weight disulfide mycothione, mycothione reductase, has led us to examine the mechanism of catalysis in greater detail. The pH dependence of the kinetic parameters V and V/K for NADPH, NADH, and an active analogue of mycothione disulfide, des-myo-inositol mycothione disulfide, has been determined. An analysis of the pH profiles has allowed the tentative assignment of catalytically significant residues crucial to the mechanism of disulfide reduction, namely, the His444-Glu449 ion pair and Cys39. Solvent kinetic isotope effects were observed on V and V/K(DIMSSM), yielding values of 1.7 +/- 0.2 and 1.4 +/- 0.2, respectively, but not on V/K(NADPH). Proton inventory studies (V versus mole fraction of D(2)O) were linear, indicative of a single proton transfer in a solvent isotopically sensitive step. Steady-state primary deuterium kinetic isotope effects on V have been determined using NADPH and NADH, yielding values of 1.27 +/- 0.03 and 1.66 +/- 0.14, respectively. The pre-steady-state primary deuterium kinetic isotope effect on enzyme reduction has values of 1.82 +/- 0.04 and 1.59 +/- 0.06 for NADPH and NADH, respectively. The steady-state primary deuterium kinetic isotope effect using NADH coincide with that obtained under single turnover conditions, suggesting the complete expression of the intrinsic primary kinetic isotope effect. Rapid reaction studies on the reductive half-reaction using NADPH and NADH yielded maximal rates of 129 +/- 2 and 20 +/- 1 s(-1), respectively, while similar studies of the oxidation of the two-electron reduced enzyme by mycothiol disulfide yielded a maximum rate of 190 +/- 10 s(-1). These data suggest a unique flavoprotein disulfide mechanism in which the rate of the oxidative half-reaction is slightly faster than the rate of the reductive half-reaction.
Patel
M P
MP
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Blanchard
J S
JS
D003903:Deuterium / Q000737:chemistry / Q000378:metabolism*; D004220:Disulfides / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D009243:NAD / Q000737:chemistry; D009249:NADP / Q000737:chemistry; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D011522:Protons; D012997:Solvents
D004220:Disulfides; D011522:Protons; D012997:Solvents; D009243:NAD; D009249:NADP; D003903:Deuterium; D010088:Oxidoreductases; C121837:mycothione reductase