Premium
Substrate specificity and the effect of calcium on Trypanosoma brucei metacaspase 2
Author(s) -
Machado Maurício F. M.,
Marcondes Marcelo F.,
Juliano Maria A.,
McLuskey Karen,
Mottram Jeremy C.,
Moss Catherine X.,
Juliano Luiz,
Oliveira Vitor
Publication year - 2013
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.12248
Subject(s) - peptide , cysteine , enzyme , active site , biochemistry , residue (chemistry) , substrate (aquarium) , trypanosoma brucei , stereochemistry , yeast , chemistry , binding site , enzyme kinetics , biology , ecology , gene
Metacaspases are cysteine peptidases found only in yeast, plants and lower eukaryotes, including the protozoa. To investigate the extended substrate specificity and effects of C a 2+ on the activation of these enzymes, detailed kinetic, biochemical and structural analyses were carried out on metacaspase 2 from T rypanosoma brucei ( T b MCA 2). These results reveal that T b MCA 2 has an unambiguous preference for basic amino acids at the P 1 position of peptide substrates and that this is most probably a result of hydrogen bonding from the P 1 residue to A sp95 and A sp211 in T b MCA 2. In addition, T b MCA 2 also has a preference for charged residues at the P 2 and P 3 positions and for small residues at the prime side of a peptide substrate. Studies into the effects of C a 2+ on the enzyme revealed the presence of two C a 2+ binding sites and a reversible structural modification of the enzyme upon C a 2+ binding. In addition, the concentration of C a 2+ used for activation of T b MCA 2 was found to produce a differential effect on the activity of T b MCA 2, but only when a series of peptides that differed in P 2 were examined, suggesting that C a 2+ activation of T b MCA 2 has a structural effect on the enzyme in the vicinity of the S 2 binding pocket. Collectively, these data give new insights into the substrate specificity and C a 2+ activation of T b MCA 2. 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.