TAILIEUCHUNG - Báo cáo khoa học: Differences in substrate specificities between cysteine protease CPB isoforms of Leishmania mexicana are mediated by a few amino acid changes

TheCPBgenes of the protozoan parasiteLeishmania mex-icana encode stage-regulated cathepsin L-like cysteine pro-teases that are important virulence factors and are in a tandem array of 19 genes. In this study, we have compared the substrate preferences of two CPB isoforms, and CPB3, and a H84Y mutant of the latter enzyme, to analyse the roles played by the few amino acid differences between the isoenzymes in determining substrate specificity. CPB3 differs at just three residues (N60D,D61Nand D64S) in the mature domain. . | Eur. J. Biochem. 271 3704-3714 2004 FEBS 2004 doi Differences in substrate specificities between cysteine protease CPB isoforms of Leishmania mexicana are mediated by a few amino acid changes Maria A. Juliano1 Darren R. Brooks2 Paul M. Selzer3 Hector L. Pandolfo1 Wagner A. S. Judice1 Luiz Juliano1 Morten Meldal4 Sanya J. Sanderson5 Jeremy C. Mottram2 and Graham H. Coombs5 1 Department of Biophysics Escola Paulista de Medicina Universidade Federal de Sao Paulo Brazil 2 Wellcome Centre for Molecular Parasitology The Anderson College University of Glasgow UK 3Akzo Nobel Intervet Innovation GmbH BioChemInformatics Schwabenheim Germany 4Center for Solid-Phase Organic Combinatorial Chemistry Department of Chemistry Carlsberg Laboratory Valby Denmark 5Division of Infection and Immunity Institute of Biomedical and Life Sciences Joseph Black Building University of Glasgow UK The CPB genes of the protozoan parasite Leishmania mex-icana encode stage-regulated cathepsin L-like cysteine proteases that are important virulence factors and are in a tandem array of 19 genes. In this study we have compared the substrate preferences of two CPB isoforms and CPB3 and a H84Y mutant of the latter enzyme to analyse the roles played by the few amino acid differences between the isoenzymes in determining substrate specificity. CPB3 differs from at just three residues N60D D61N and D64S in the mature domain. The H84Y mutation mimics an additional change present in another isoenzyme CPB18. The active recombinant CPB isoenzymes and mutant were produced using Escherichia coli and the S1-S3 and S -S subsite specificities determined using a series of fluorogenic peptide derivatives in which substitutions were made on positions P3 to P3 by natural amino acids. Carboxydipeptidase activities of CPB3 and H84Y were also observed using the peptide Abz-FRAK Dnp -OH and some of its analogues. The kinetic parameters of hydrolysis by CPB3 H84Y and of .

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