Specific negative charges in cysteine protease isoforms of Leishmania mexicana are highly influential on the substrate binding and hydrolysis

Specific negative charges in cysteine protease isoforms of Leishmania mexicana are highly influential on the substrate binding and hydrolysis

Autor Judice, WAS Google Scholar
Mottram, J. C. Google Scholar
Coombs, G. H. Google Scholar
Juliano, M. A. Google Scholar
Juliano, L. Google Scholar
Instituição Universidade Federal de São Paulo (UNIFESP)
Univ Glasgow
Resumo We focused on the importance of the electrostatic environment on the catalytic properties of the Leishmania mexicana CPB recombinant isoenzymes (rCPB2.8, rCPB3 and its mutant rH84Y), by investigating the influence of pH and NaCl on their hydrolytic activities. rCPB2.8 contains the residues Asn60, Asp61 and Asp64; rCPB3 presents the three variant residues Asp60, Asn61 and Ser-64 and the mutant of the latter isoform, rH84Y, has a mutation on the outer loop residue (His84 to Tyr). Synthetic fluorescence resonance energy transfer (FRET) peptides, which contain different positive charge distribution in their sequences were used as substrates. the results show that hydrolytic efficiency is dependent of the positive charge distribution in the substrates and that NaCl activated rCPB2.8 and rCPB3 in acidic pH but inhibited them at pH higher than 5. the rate constants of substrate diffusion (k(1)), substrate dissociation (k(-1)), acylation (k(2)) and deacylation (k(3)) and the corresponding activation energies and entropies were derived. Significant differences in the kinetic rate constants (k) and entropies were found between the CPB isoforms, and the diffusion process seems to be the limiting step. the activation energy of denaturation (Ea-Den) and entropy of denaturation (Delta S-Den) of rCPB3 were higher than those for rCPB2.8, suggesting higher salvation and protein structure for rCPB3. Thus the findings suggest that the two CPB isoenzymes with a few negative charge modifications provide the parasite with an array of hydrolytic activity and enzymatic adaptation to pH, salinity and temperature that may be needed for its interaction with the mammalian host. (c) 2005 Elsevier B.V. All rights reserved.
Palavra-chave Leishmania mexicana
cysteine protease
fluorogenic
FRET peptides
Idioma Inglês
Data de publicação 2005-11-01
Publicado em Molecular and Biochemical Parasitology. Amsterdam: Elsevier B.V., v. 144, n. 1, p. 36-43, 2005.
ISSN 0166-6851 (Sherpa/Romeo, fator de impacto)
Publicador Elsevier B.V.
Extensão 36-43
Fonte http://dx.doi.org/10.1016/j.molbiopara.2005.07.004
Direito de acesso Acesso restrito
Tipo Artigo
Web of Science WOS:000232847300005
Endereço permanente http://repositorio.unifesp.br/handle/11600/28529

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