Substrate specificity and the effect of calcium on Trypanosomabrucei 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 Ca2+ on the activation of these enzymes, detailed kinetic, biochemical and structural analyses were carried out on metacaspase 2 from Trypanosomabrucei (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 Ca2+ on the enzyme revealed the presence of two Ca2+ binding sites and a reversible structural modification of the enzyme upon Ca2+ binding. in addition, the concentration of Ca2+ 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 Ca2+ 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 Ca2+ 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.