Characterization of a cysteine protease from bloodstream forms of Trypanosoma congolense

• Published in: European journal of biochemistry Vol. 204; no. 1; pp. 371 - 379
• Main Authors: Mbawa, Z.R. (International Laboratory for Research on Animal Disease, Nairobi, Kenya), Gumm, I.D, Shaw, E, Lonsdale-Eccles, J.D
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01.02.1992; Blackwell
• Subjects: ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Chromatography, Gel; Concanavalin A - metabolism; Coumarins - metabolism; Cysteine Endopeptidases - chemistry; Cysteine Endopeptidases - metabolism; Diazomethane - metabolism; Electrophoresis, Polyacrylamide Gel; Enzyme Activation - drug effects; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Hydrolases; Isoelectric Point; Kinetics; Molecular Sequence Data; Molecular Weight; Peptides - chemistry; Peptides - metabolism; PROPIEDADES FISICO-QUIMICAS; PROPRIETE PHYSICOCHIMIQUE; Protease Inhibitors - pharmacology; PURIFICACION; PURIFICATION; Rats; Rats, Inbred Strains; Substrate Specificity; Sulfhydryl Compounds - pharmacology; TRYPANOSOMA CONGOLENSE; Trypanosoma congolense - enzymology; Trypanosomiasis, African - parasitology; Variant Surface Glycoproteins, Trypanosoma - metabolism; Protozoa; Cathepsin L; Purification; Enzyme; Cysteine proteinase; Trypanosoma congolense; Rhizoflagellata; Enzyme inhibitor; Kinetic parameter; Physicochemical properties
• ISSN: 0014-2956; 1432-1033
• DOI: 10.1111/j.1432-1033.1992.tb16646.x

A cysteine protease (trypanopain-Tc) with cathepsin-L-like properties has been purified from Trypanosoma congolense. The enzyme has an apparent molecular mass of 31 - 32 kDa by SDS/PAGE and 66 kDa by gel chromatography. It has a pI 7.4 and a high affinity for concanavalin A. Trypanopain-Tc catalyses the limited proteolysis of a variety of protein substrates such as fibrinogen, serum albumin and trypanosome variant-surface glycoprotein. It has minimal or no activity against casein or elastin. A variety of peptidyl amidomethylcoumarins and peptidyl diazomethanes were used to test the specificity of trypanopain-Tc. The better substrates had Arg or Lys in P1 and hydrophobic amino acids in P2 and P3. The best substrate found for trypanopain-Tc was Z-Phe-Arg-NHMec (Z, benzyl-oxycarbonyl; NHMec, 7-amido-4-methylcoumarin). The kinetic constants for the hydrolysis of Z-Phe-Arg-NHMec were kcat = 17.4 s-1, Km = 4.4 micromole, kcat/Km = 4.0 micromole-1.S-1, which are very similar to those of cathepsin L with this substrate. The specific substrates for cathepsin B (Z-Arg-Arg-NHMec) and cathepsin H (Arg-NHMec) were not hydrolysed by trypanopain-Tc under the conditions tested. The pH optimum of trypanopain-tc against Z-Hhe-Arg-NHMec was pH 6.0 but it showed a broad peak of activity extending well into the alkaline region. The enzyme was activated by low-molecular-mass thiol compounds and inhibited by cystatin, L-trans-epoxysuccinyl-4-guanidinobutane (E-64) and a variety of peptidyl diazomethanes. The most effective diazomethane inhibitors (Z-Leu-Leu-Met-CHN2, Z-Leu-Met-CHN2 and Z-Leu-Lye-CHN2, were inhibitory at nanomolar concentrations and were trypanocidal in vitro after 24 - 48 h incubation in 20 micromole [inhibitor]. However, it is not clear whether the trypanocidal activity of these inhibitors is a consequence of the inhibition of trypanopains or of some other essential proteolytic activities within the parasites