Substrate specificity and the effect of calcium on Trypanosomabrucei metacaspase 2

Substrate specificity and the effect of calcium on Trypanosomabrucei metacaspase 2

Author Machado, Mauricio F. M. Autor UNIFESP Google Scholar
Marcondes, Marcelo F. Autor UNIFESP Google Scholar
Juliano, Maria A. Autor UNIFESP Google Scholar
McLuskey, Karen Google Scholar
Mottram, Jeremy C. Google Scholar
Moss, Catherine X. Google Scholar
Juliano, Luiz Autor UNIFESP Google Scholar
Oliveira, Vitor Autor UNIFESP Google Scholar
Institution Universidade Federal de São Paulo (UNIFESP)
Univ Glasgow
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.
Keywords calcium binding
kinetic parameters
structural modification
substrate specificity
Language English
Sponsor Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Wellcome Trust
Medical Research Council
Grant number FAPESP: 2008/57336-2
FAPESP: 2011/51718-3
CNPq: 470044/2010-1
Wellcome Trust: 091790
Wellcome Trust: 085349
Medical Research Council: 0700127
Date 2013-06-01
Published in Febs Journal. Hoboken: Wiley-Blackwell, v. 280, n. 11, p. 2608-2621, 2013.
ISSN 1742-464X (Sherpa/Romeo, impact factor)
Publisher Wiley-Blackwell
Extent 2608-2621
Access rights Open access Open Access
Type Article
Web of Science ID WOS:000319413400006

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