Mechanisms of allosteric regulation of trypanosoma cruzi S-adenosylmethionine decarboxylase

Tracy Clyne Beswick, Erin K. Willert, Margaret A. Phillips

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

S-Adenosylmethionine decarboxylase (AdoMetDC) is a pyruvoyl-dependent enzyme that catalyzes an essential step in polyamine biosynthesis. The polyamines are required for cell growth, and the biosynthetic enzymes are targets for antiproliferative drugs. The function of AdoMetDC is regulated by the polyamine-precursor putrescine in a species-specific manner. AdoMetDC from the protozoal parasite Trypanosoma cruzi requires putrescine for maximal enzyme activity, but not for processing to generate the pyruvoyl cofactor. The putrescine-binding site is distant from the active site, suggesting a mechanism of allosteric regulation. To probe the structural basis by which putrescine stimulates T. cruzi AdoMetDC we generated mutations in both the putrescine-binding site and the enzyme active site. The catalytic efficiency of the mutant enzymes, and the binding of the diamidine inhibitors, CGP 48664A and CGP 40215, were analyzed. Putrescine stimulates the kcat/K m for wild-type T. cruzi AdoMetDC by 27-fold, and it stimulates the binding of both inhibitors (IC50s decrease 10-20-fold with putrescine). Unexpectedly CGP 48664A activated the T. cruzi enzyme at low concentrations (0.1-10 μM), while at higher concentrations (>100 μM), or in the presence of putrescine, inhibition was observed. Analysis of the mutant data suggests that this inhibitor binds both the putrescine-binding site and the active site, providing evidence that the putrescine-binding site of the T. cruzi enzyme has broad ligand specificity. Mutagenesis of the active site identified residues that are important for putrescine stimulation of activity (F7 and T245), while none of the active site mutations altered the apparent putrescine-binding constant. Mutations of residues in the putrescine-binding site that resulted in reduced (S111R) and enhanced (F285H) catalytic efficiency were both identified. These data provide evidence for coupling between residues in the putrescine-binding site and the active site, consistent with a mechanism of allosteric regulation.

Original languageEnglish (US)
Pages (from-to)7797-7807
Number of pages11
JournalBiochemistry
Volume45
Issue number25
DOIs
StatePublished - Jun 27 2006

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Adenosylmethionine Decarboxylase
Allosteric Regulation
Putrescine
Trypanosoma cruzi
Catalytic Domain
4-amidinoindan-1-one 2'-amidinohydrazone
Binding Sites
Enzymes
Polyamines
Mutation
Pentamidine
Mutagenesis
Biosynthesis
Enzyme activity
Cell growth

ASJC Scopus subject areas

  • Biochemistry

Cite this

Mechanisms of allosteric regulation of trypanosoma cruzi S-adenosylmethionine decarboxylase. / Beswick, Tracy Clyne; Willert, Erin K.; Phillips, Margaret A.

In: Biochemistry, Vol. 45, No. 25, 27.06.2006, p. 7797-7807.

Research output: Contribution to journalArticle

Beswick, Tracy Clyne ; Willert, Erin K. ; Phillips, Margaret A. / Mechanisms of allosteric regulation of trypanosoma cruzi S-adenosylmethionine decarboxylase. In: Biochemistry. 2006 ; Vol. 45, No. 25. pp. 7797-7807.
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abstract = "S-Adenosylmethionine decarboxylase (AdoMetDC) is a pyruvoyl-dependent enzyme that catalyzes an essential step in polyamine biosynthesis. The polyamines are required for cell growth, and the biosynthetic enzymes are targets for antiproliferative drugs. The function of AdoMetDC is regulated by the polyamine-precursor putrescine in a species-specific manner. AdoMetDC from the protozoal parasite Trypanosoma cruzi requires putrescine for maximal enzyme activity, but not for processing to generate the pyruvoyl cofactor. The putrescine-binding site is distant from the active site, suggesting a mechanism of allosteric regulation. To probe the structural basis by which putrescine stimulates T. cruzi AdoMetDC we generated mutations in both the putrescine-binding site and the enzyme active site. The catalytic efficiency of the mutant enzymes, and the binding of the diamidine inhibitors, CGP 48664A and CGP 40215, were analyzed. Putrescine stimulates the kcat/K m for wild-type T. cruzi AdoMetDC by 27-fold, and it stimulates the binding of both inhibitors (IC50s decrease 10-20-fold with putrescine). Unexpectedly CGP 48664A activated the T. cruzi enzyme at low concentrations (0.1-10 μM), while at higher concentrations (>100 μM), or in the presence of putrescine, inhibition was observed. Analysis of the mutant data suggests that this inhibitor binds both the putrescine-binding site and the active site, providing evidence that the putrescine-binding site of the T. cruzi enzyme has broad ligand specificity. Mutagenesis of the active site identified residues that are important for putrescine stimulation of activity (F7 and T245), while none of the active site mutations altered the apparent putrescine-binding constant. Mutations of residues in the putrescine-binding site that resulted in reduced (S111R) and enhanced (F285H) catalytic efficiency were both identified. These data provide evidence for coupling between residues in the putrescine-binding site and the active site, consistent with a mechanism of allosteric regulation.",
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N2 - S-Adenosylmethionine decarboxylase (AdoMetDC) is a pyruvoyl-dependent enzyme that catalyzes an essential step in polyamine biosynthesis. The polyamines are required for cell growth, and the biosynthetic enzymes are targets for antiproliferative drugs. The function of AdoMetDC is regulated by the polyamine-precursor putrescine in a species-specific manner. AdoMetDC from the protozoal parasite Trypanosoma cruzi requires putrescine for maximal enzyme activity, but not for processing to generate the pyruvoyl cofactor. The putrescine-binding site is distant from the active site, suggesting a mechanism of allosteric regulation. To probe the structural basis by which putrescine stimulates T. cruzi AdoMetDC we generated mutations in both the putrescine-binding site and the enzyme active site. The catalytic efficiency of the mutant enzymes, and the binding of the diamidine inhibitors, CGP 48664A and CGP 40215, were analyzed. Putrescine stimulates the kcat/K m for wild-type T. cruzi AdoMetDC by 27-fold, and it stimulates the binding of both inhibitors (IC50s decrease 10-20-fold with putrescine). Unexpectedly CGP 48664A activated the T. cruzi enzyme at low concentrations (0.1-10 μM), while at higher concentrations (>100 μM), or in the presence of putrescine, inhibition was observed. Analysis of the mutant data suggests that this inhibitor binds both the putrescine-binding site and the active site, providing evidence that the putrescine-binding site of the T. cruzi enzyme has broad ligand specificity. Mutagenesis of the active site identified residues that are important for putrescine stimulation of activity (F7 and T245), while none of the active site mutations altered the apparent putrescine-binding constant. Mutations of residues in the putrescine-binding site that resulted in reduced (S111R) and enhanced (F285H) catalytic efficiency were both identified. These data provide evidence for coupling between residues in the putrescine-binding site and the active site, consistent with a mechanism of allosteric regulation.

AB - S-Adenosylmethionine decarboxylase (AdoMetDC) is a pyruvoyl-dependent enzyme that catalyzes an essential step in polyamine biosynthesis. The polyamines are required for cell growth, and the biosynthetic enzymes are targets for antiproliferative drugs. The function of AdoMetDC is regulated by the polyamine-precursor putrescine in a species-specific manner. AdoMetDC from the protozoal parasite Trypanosoma cruzi requires putrescine for maximal enzyme activity, but not for processing to generate the pyruvoyl cofactor. The putrescine-binding site is distant from the active site, suggesting a mechanism of allosteric regulation. To probe the structural basis by which putrescine stimulates T. cruzi AdoMetDC we generated mutations in both the putrescine-binding site and the enzyme active site. The catalytic efficiency of the mutant enzymes, and the binding of the diamidine inhibitors, CGP 48664A and CGP 40215, were analyzed. Putrescine stimulates the kcat/K m for wild-type T. cruzi AdoMetDC by 27-fold, and it stimulates the binding of both inhibitors (IC50s decrease 10-20-fold with putrescine). Unexpectedly CGP 48664A activated the T. cruzi enzyme at low concentrations (0.1-10 μM), while at higher concentrations (>100 μM), or in the presence of putrescine, inhibition was observed. Analysis of the mutant data suggests that this inhibitor binds both the putrescine-binding site and the active site, providing evidence that the putrescine-binding site of the T. cruzi enzyme has broad ligand specificity. Mutagenesis of the active site identified residues that are important for putrescine stimulation of activity (F7 and T245), while none of the active site mutations altered the apparent putrescine-binding constant. Mutations of residues in the putrescine-binding site that resulted in reduced (S111R) and enhanced (F285H) catalytic efficiency were both identified. These data provide evidence for coupling between residues in the putrescine-binding site and the active site, consistent with a mechanism of allosteric regulation.

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