title: The crystal structure of Trypanosoma brucei enolase: visualisation of the inhibitory metal binding site III and potential as target for selective, irreversible inhibition.
abstract: The glycolytic enzymes of the trypanosomatids, that cause a variety of medically and agriculturally important diseases, are validated targets for drug design. Design of species-specific inhibitors is facilitated by the availability of structural data. Irreversible inhibitors, that bound covalently to the parasite enzyme alone, would be potentially particularly effective. Here we determine the crystal structure of enolase from Trypanosoma brucei and show that two cysteine residues, located in a water-filled cavity near the active-site, are modified by iodoacetamide leading to loss of catalytic activity. Since these residues are specific to the Trypanosomatidae lineage, this finding opens the way for the development of parasite-specific, irreversibly binding enolase inhibitors. In the present structure, the catalytic site is partially occupied by sulphate and two zinc ions. Surprisingly, one of these zinc ions illustrates the existence of a novel enolase-binding site for divalent metals. Evidence suggests that this is the first direct visualization of the elusive inhibitory metal site, whose existence has hitherto only been inferred from kinetic data.
authors.affiliation: Instituto de Física de São Carlos, Universidade de São Paulo, São Carlos SP, Brazil.
authors.forename: Maria Teresa
authors.initials: MT
authors.lastname: da Silva Giotto
authors.forename: Véronique
authors.initials: V
authors.lastname: Hannaert
authors.forename: Didier
authors.initials: D
authors.lastname: Vertommen
authors.forename: Marcos V
authors.initials: MV
authors.lastname: de A S Navarro
authors.forename: Mark H
authors.initials: MH
authors.lastname: Rider
authors.forename: Paul A M
authors.initials: PA
authors.lastname: Michels
authors.forename: Richard C
authors.initials: RC
authors.lastname: Garratt
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Rigden
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D007460:Iodoacetamide / Q000737:chemistry; D008024:Ligands; D008670:Metals / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010751:Phosphopyruvate Hydratase / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D021241:Spectrometry, Mass, Electrospray Ionization; D013431:Sulfates / Q000378:metabolism; D014346:Trypanosoma brucei brucei / Q000201:enzymology*; D015032:Zinc / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D008024:Ligands; D008670:Metals; D013431:Sulfates; D010751:Phosphopyruvate Hydratase; D015032:Zinc; D007460:Iodoacetamide