Cloning, expression, characterization and inhibition studies on trypanothione synthetase, a drug target enzyme, from Leishmania donovani

2011 ◽  
Vol 392 (12) ◽  
pp. 1113-1122 ◽  
Author(s):  
Prakash Saudagar ◽  
Vikash Kumar Dubey
Keyword(s):  
Gene Cloning ◽  
Drug Target ◽  
Leishmania Donovani ◽  
Redox Homeostasis ◽  
Substrate Inhibition ◽  
Leishmanicidal Activity ◽  
High Substrate ◽  
Glutathione Concentration ◽  
Expression Characterization ◽  
Inhibition Studies

Abstract Trypanothione synthetase, a validated drug target, synthesizes trypanothione from glutathione and spermidine. Here we report the gene cloning, expression, characterization and inhibition studies of trypanothione synthetase from Leishmania donovani (LdTryS). The purified recombinant LdTryS enzyme obeyed Michaelis-Menten kinetics. High substrate inhibition was observed with glutathione (Km=33.24 μm, kcat=1.3 s-1, Ki=866 μm). The enzyme shows simple hyperbolic kinetics with fixed glutathione concentration and with other substrates limiting Km values for Mg. ATP and spermidine of 14.2 μm and 139.6 μm, respectively. LdTryS was also screened for inhibitors. Tomatine, conessine, uvaol and betulin were identified as inhibitors of the enzyme and were tested for leishmanicidal activity. Finally, the effect of LdTryS inhibitors on redox homeostasis of the parasite gives a broader picture of their action against leishmaniasis.

In vitro and in vivo leishmanicidal activity of Dysoxylum binectariferum and its fractions against Leishmania donovani

Phytomedicine ◽  
2007 ◽  
Vol 14 (1) ◽  
pp. 36-42 ◽  
Author(s):  
V. Lakshmi ◽  
K. Pandey ◽  
A. Kapil ◽  
N. Singh ◽  
M. Samant ◽  
...  
Keyword(s):  
Leishmania Donovani ◽  
Leishmanicidal Activity ◽  
Dysoxylum Binectariferum

scholarly journals Purification and kinetic properties of ox brain histamine N-methyltransferase

1986 ◽  
Vol 233 (3) ◽  
pp. 669-676 ◽  
Author(s):  
W L Gitomer ◽  
K F Tipton
Keyword(s):  
Acetic Acid ◽  
Steady State ◽  
Mouse Brain ◽  
Initial Rate ◽  
Substrate Inhibition ◽  
Gel Filtration ◽  
Product Inhibition ◽  
Kinetic Properties ◽  
Brain Histamine ◽  
Inhibition Studies

Histamine N-methyltransferase (EC 2.1.1.8) was purified 1100-fold from ox brain. The native enzyme has an Mr of 34800 +/- 2400 as measured by gel filtration on Sephadex G-100. The enzyme is highly specific for histamine. It does not methylate noradrenaline, adrenaline, DL-3,4-dihydroxymandelic acid, 3,4-dihydroxyphenylacetic acid, 3-hydroxytyramine or imidazole-4-acetic acid. Unlike the enzyme from rat and mouse brain, ox brain histamine N-methyltransferase did not exhibit substrate inhibition by histamine. Initial rate and product inhibition studies were consistent with an ordered steady-state mechanism with S-adenosylmethionine being the first substrate to bind to the enzyme and N-methylhistamine being the first product to dissociate.

scholarly journals Time-dependent activation of the semicarbazide-sensitive amine oxidase (SSAO) from ox lung microsomes

2000 ◽  
Vol 351 (3) ◽  
pp. 789-794 ◽  
Author(s):  
Jose M. LIZCANO ◽  
Keith F. TIPTON ◽  
Mercedes UNZETA
Keyword(s):  
Amine Oxidase ◽  
Reaction Pathway ◽  
Substrate Inhibition ◽  
Kinetic Studies ◽  
Tryptophan Fluorescence ◽  
The Other ◽  
Time Dependent ◽  
High Substrate ◽  
Before And After ◽  
Lag Period

The activity of ox lung microsomal semicarbazide-sensitive amine oxidase (EC 1.4.3.6; SSAO) towards benzylamine increased 20-fold during incubation at 37°C. After an initial lag-period, activation was first-order with time and complete after approx. 20h. No significant changes in activity towards methylamine, histamine or 2-phenylethylamine were observed, although mixed-substrate experiments were consistent with the same enzyme being involved in the oxidation of all these substrates, both before and after time-dependent activation. The enzyme-tryptophan fluorescence increased on incubation at 37°C in parallel with the increase in activity towards benzylamine. Treatment of the activated-enzyme preparation with 6M guanidinium chloride followed by dialysis, caused both the activity towards benzylamine and the fluorescence to fall to that occurring before activation. However, incubation of this preparation at 37°C resulted in increases in fluorescence and activity similar to those seen with the unactivated enzyme. Benzylamine oxidation was inhibited, uncompetitively with respect to oxygen, by high substrate concentrations but no such inhibition was observed with the other amines. Activation resulted in an increase in Vmax for benzylamine oxidation, with no significant alterations in the Km or the Ksi for high-substrate inhibition. Kinetic studies were consistent with sequential mechanisms being followed for the oxidation of both benzylamine and methylamine but the dependence on oxygen concentration was complex. These results might indicate that benzylamine follows a different reaction pathway from the other substrates, with substrate-specific activation involving a reaction step that is rate-limiting for benzylamine oxidation but not for the others.

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