Characterization of an endopeptidase of Trypanosoma brucei brucei.
A soluble 80-kDa endopeptidase has been isolated from Trypanosoma brucei brucei. The enzyme, which has a pI 5.1, is optimally active at about pH 8.2 and has apparent pKa values of 6.0 and greater than or equal to 10. It is inhibited by the serine protease inhibitor diisopropylfluorophosphate and by the serine protease mechanism-based inhibitor 3,4-dichloroisocoumarin. Unexpectedly, the enzyme is inhibited by the cysteine protease inhibitor benzyloxycarbonyl-Leu-Lys-CHN2 but not by the related diazomethane, butoxycarbonyl-Val-Leu-Gly-Lys-CHN2, nor by other cysteine protease specific compounds. Specificity studies with a variety of amidomethylcoumaryl (AMC) derivatives of small peptides show that the enzyme has a highly restricted trypsin-like specificity. The best substrate, based on the magnitude of kcat/Km, was benzyloxycarbonyl-Arg-Arg-AMC; other good substrates were benzyloxycarbonyl-Phe-Arg-AMC, benzoyl-Arg-AMC, and compounds with Arg at P1 and Ala or Gly at P2. The hydrolysis of most substrates obeyed classical Michaelis-Menton kinetics but several exhibited pronounced substrate inhibition. The enzyme did not activate plasminogen nor decrease blood clotting time; it was inhibited by aprotinin but not by chicken ovomucoid. We conclude that the enzyme is a trypsin-like serine endopeptidase with unusually restricted subsite specificities.
Kornblatt
M J
MJ
Department of Chemistry and Biochemistry, Concordia University, Montreal, Quebec, Canada.
Mpimbaza
G W
GW
Lonsdale-Eccles
J D
JD
D000595:Amino Acid Sequence; D000818:Animals; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007531:Isoflurophate / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D012995:Solubility; D013379:Substrate Specificity; D014346:Trypanosoma brucei brucei / Q000201:enzymology*
D010455:Peptides; D011480:Protease Inhibitors; D007531:Isoflurophate; D010450:Endopeptidases