S-Adenosylmethionine synthetase in bloodstream Trypanosoma brucei |
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Authors: | Nigel Yarlett Joanne Garofalo Burt Goldberg Mary Ann Ciminelli Vincent Ruggiero Janice R Sufrin Cyrus J Bacchi |
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Institution: | 1. Haskins Laboratories and Biology Department, Pace University, New York, NY, USA;2. Grace Cancer Drug Center Roswell Park Cancer Institute, Buffalo, NY, USA |
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Abstract: | S-adenosylmethionine synthetase was studied from bloodstream forms of Trypanosoma brucei brucei, the agent of African sleeping sickness. Two isoforms of the enzyme were evident from Eadie Hofstee and Hanes-Woolf plots of varying ATP or methionine concentrations. In the range 10–250 μM the Km for methionine was 20 μM, and this changed to 200 μM for the range 0.5–5.0 mM. In the range 10–250 μM the Km for ATP was 53 μM, and this changed to 1.75 mM for the range 0.5–5.0 mM. The trypanosome enzyme had a molecular weight of 145 kDa determined by agarose gel filtration. Methionine analogs including selenomethionine, L-2-amino-4-methoxy-cis but-3-enoic acid and ethionine acted as competitive inhibitors of methionine and as weak substrates when tested in the absence of methionine with 14C]ATP. The enzyme was not inducible in procyclic trypomastigotes in vitro, and the enzyme half-life was > 6 h. T. b. brucei AdoMet synthetase was inhibited by AdoMet (Ki 240 μM). The relative insensitivity of the trypanosome enzyme to control by product inhibition indicates it is markedly different from mammalian isoforms of the enzyme which are highly sensitive to AdoMet. Since trypanosomes treated with the ornithine decarboxylase antagonist DL-α-difluoromethylornithine accumulate AdoMet and dcAdoMet (final concentration ≈ 5 mM), this enzyme may be the critical drug target linking inhibition of polyamine synthesis to disruption of AdoMet metabolism. |
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Keywords: | S-Adenosylmethionine synthetase S-Adenosylmethionine Polyamine metabolism Methionine analog AdoMet AdoHcy S-adenosyl-homocysteine dcAdoMet SDS Na dodecyl sulfate DFMO HEPES |
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