A COMPARISON OF THE ENZYMATIC-PROPERTIES OF THE MAJOR CYSTEINE PROTEINASES FROM TRYPANOSOMA-CONGOLENSE AND TRYPANOSOMA-CRUZI

Congopain and cruzipain, the major cysteine proteinases from Trypanoso ma congolense and Trypanosoma cruzi, were compared for their activitie s towards a series of new, sensitive fluorogenic substrates of the pap ain family of cysteine proteinases and far their sensitivity to inhibi tion by cystatins and related biotinylated peptidyl diazomethanes. Low K-i values, in the 10 pM range, were found for the interaction of bot h proteinases with natural cystatin inhibitors. The kinetic constants for the hydrolysis of cystatin-derived substrates, and the inhibition by related diazomethanes were essentially identical. Unlike cathepsins B and L, the related mammal papain family proteinases, congopain and cruzipain accomodate a prolyl residue in P2'. Substrates having the se quence VGGP from P2 to P2' were hydrolysed by both congopain and cruzi pain with a k(cat)/K-m greater than 4.10(3) mM(-1) s(-1). Irreversible diazomethane inhibitors, deduced from the unprime sequence of cystati n-derived substrates, inhibited the two parasite proteinases. N-termin al labelling of diazomethanes with a biotin group did not alter the ra te of inhibition significantly, which provides a useful tool for exami ning the distribution of these enzymes in the parasite and in the host . Despite their similar activities on cystatin-derived substrates, con gopain and cruzipain had significantly different pH-activity profiles when assayed with a cystatin-derived substrate. They were correlated w ith structural differences, especially at the presumed S2 subsites. (C ) 1997 Elsevier Science B.V.