title: Substrate specificity and the effect of calcium on Trypanosoma brucei metacaspase 2.
abstract: Metacaspases are cysteine peptidases found only in yeast, plants and lower eukaryotes, including the protozoa. To investigate the extended substrate specificity and effects of Ca(2+) on the activation of these enzymes, detailed kinetic, biochemical and structural analyses were carried out on metacaspase 2 from Trypanosoma brucei (TbMCA2). These results reveal that TbMCA2 has an unambiguous preference for basic amino acids at the P1 position of peptide substrates and that this is most probably a result of hydrogen bonding from the P1 residue to Asp95 and Asp211 in TbMCA2. In addition, TbMCA2 also has a preference for charged residues at the P2 and P3 positions and for small residues at the prime side of a peptide substrate. Studies into the effects of Ca(2+) on the enzyme revealed the presence of two Ca(2+) binding sites and a reversible structural modification of the enzyme upon Ca(2+) binding. In addition, the concentration of Ca(2+) used for activation of TbMCA2 was found to produce a differential effect on the activity of TbMCA2, but only when a series of peptides that differed in P2 were examined, suggesting that Ca(2+) activation of TbMCA2 has a structural effect on the enzyme in the vicinity of the S2 binding pocket. Collectively, these data give new insights into the substrate specificity and Ca(2+) activation of TbMCA2. This provides important functional details and leads to a better understanding of metacaspases, which are known to play an important role in trypanosomes and make attractive drug targets due to their absence in humans.
authors.affiliation: Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo 04039-032, Brazil.
authors.forename: Maurício F M
authors.initials: MF
authors.lastname: Machado
authors.forename: Marcelo F
authors.initials: MF
authors.lastname: Marcondes
authors.forename: Maria A
authors.initials: MA
authors.lastname: Juliano
authors.forename: Karen
authors.initials: K
authors.lastname: McLuskey
authors.forename: Jeremy C
authors.initials: JC
authors.lastname: Mottram
authors.forename: Catherine X
authors.initials: CX
authors.lastname: Moss
authors.forename: Luiz
authors.initials: L
authors.lastname: Juliano
authors.forename: Vitor
authors.initials: V
authors.lastname: Oliveira
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D020013:Calcium Signaling / Q000235:genetics / Q000502:physiology*; D018360:Crystallography, X-Ray; D057056:Cysteine Proteases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity / Q000235:genetics; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics
chemical_list: D015800:Protozoan Proteins; D057056:Cysteine Proteases