Characterisation of a cysteine protease from bloodstream forms of Trypanosoma congolense.
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, benzyloxycarbonyl; 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 microM, kcat/Km = 4.0 microM-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-Phe-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-Lys-CHN2, were inhibitory at nanomolar concentrations and were trypanocidal in vitro after 24-48 h incubation in greater than or equal to 20 microM [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.
Mbawa
Z R
ZR
International Laboratory for Research on Animal Diseases, Nairobi, Kenya.
Gumm
I D
ID
Shaw
E
E
Lonsdale-Eccles
J D
JD
D000595:Amino Acid Sequence; D000818:Animals; D002850:Chromatography, Gel; D003208:Concanavalin A / Q000378:metabolism; D003374:Coumarins / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D003978:Diazomethane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D007526:Isoelectric Point; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D014348:Trypanosoma congolense / Q000201:enzymology*; D014353:Trypanosomiasis, African / Q000469:parasitology; D014643:Variant Surface Glycoproteins, Trypanosoma / Q000378:metabolism
D003374:Coumarins; D010455:Peptides; D011480:Protease Inhibitors; D013438:Sulfhydryl Compounds; D014643:Variant Surface Glycoproteins, Trypanosoma; D003208:Concanavalin A; D003978:Diazomethane; D003546:Cysteine Endopeptidases