title: Specific negative charges in cysteine protease isoforms of Leishmaniamexicana are highly influential on the substrate binding and hydrolysis.
abstract: 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 Ser64 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 (k1), substrate dissociation (k-1), acylation (k2) and deacylation (k3) 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 (DeltaSDen) 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.
authors.affiliation: Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Tres de Maio 100, 04044-20 São Paulo, Brazil.
authors.forename: Wagner A S
authors.initials: WA
authors.lastname: Judice
authors.forename: Jeremy C
authors.initials: JC
authors.lastname: Mottram
authors.forename: Graham H
authors.initials: GH
authors.lastname: Coombs
authors.forename: Maria A
authors.initials: MA
authors.lastname: Juliano
authors.forename: Luiz
authors.initials: L
authors.lastname: Juliano
mesh_terms: D000818:Animals; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D007894:Leishmania mexicana / Q000201:enzymology*; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D015800:Protozoan Proteins / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012965:Sodium Chloride; D013379:Substrate Specificity
chemical_list: D007527:Isoenzymes; D010455:Peptides; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D012965:Sodium Chloride; D003546:Cysteine Endopeptidases