title: Substrate inhibition and allosteric regulation by heparan sulfate of Trypanosoma brucei cathepsin L.
abstract: The cysteine protease brucipain is an important drug target in the protozoan Trypanosoma brucei, the causative agent of both Human African trypanosomiasis and Animal African trypanosomiasis. Brucipain is closely related to mammalian cathepsin L and currently used as a framework for the development of inhibitors that display anti-parasitic activity. We show that recombinant brucipain lacking the C-terminal extension undergoes inhibition by the substrate benzyloxycarbonyl-FR-7-amino-4-methylcoumarin at concentrations above the K(m), but not by benzyloxycarbonyl-VLR-7-amino-4-methylcoumarin. The allosteric modulation exerted by the substrate is controlled by temperature, being apparent at 25°C but concealed at 37°C. The behavior of the enzyme in vitro can be explained by discrete conformational changes caused by the shifts in temperature that render it less susceptible to substrate inhibition. Enzyme inhibition by the di-peptydyl substrate impaired the degradation of human fibrinogen at 25°C, but not at 37°C. We also found that heparan sulfate acts as a natural allosteric modulator of the enzyme through interactions that prevent substrate inhibition. We propose that brucipain shifts between an active and an inactive form as a result of temperature-dependent allosteric regulation.
authors.affiliation: Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Tatiana F R
authors.initials: TF
authors.lastname: Costa
authors.forename: Flavia C G
authors.initials: FC
authors.lastname: dos Reis
authors.forename: Ana Paula C A
authors.initials: AP
authors.lastname: Lima
mesh_terms: D000494:Allosteric Regulation; D000818:Animals; D056668:Cathepsin L / Q000737:chemistry* / Q000378:metabolism; D018929:Cell Culture Techniques; D003374:Coumarins / Q000737:chemistry*; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D005340:Fibrinogen / Q000378:metabolism; D006497:Heparan Sulfate / Q000737:chemistry* / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008018:Life Cycle Stages / Q000187:drug effects; D059748:Proteolysis; D015800:Protozoan Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D014346:Trypanosoma brucei brucei / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development
chemical_list: D056668:Cathepsin L; D003374:Coumarins; D003546:Cysteine Endopeptidases; D015853:Cysteine Proteinase Inhibitors; D005340:Fibrinogen; D006497:Heparan Sulfate; D015800:Protozoan Proteins; D011994:Recombinant Proteins; C100684:trypanopain Tb