Purification and characterisation of a trypsin-like serine oligopeptidase from Trypanosoma congolense

Trypanosoma brucei contain a serine oligopeptidase (OP-Tb) that is released into (and remains active in) the blood of trypanosome-infected animals. He re a similar enzyme from Trypanosoma congolense is described. This oligopep tidase, called OP-Tc, was purified using three-phase partitioning, and ion- exchange and affinity chromatography. OP-Tc is inhibited by alkylating agen ts, by serine peptidase-specific inhibitors including 3,4-dichloroisocoumar in, 4-(2-aminoethyl)benzenesulfonylfluoride and diispropylfluoro-phosphate and by other peptidase inhibitors including leupeptin, antipain and peptidy l chloromethyl ketones. Reducing agents such as dithiothreitol enhanced act ivity as did heparin, spermine and spermidine. The enzyme has trypsin-like specificity since it cleaved fluorogenic peptides that have basic amino aci d residues (Arg or Lys) in the P-1 position. Potential substrates without a basic residue in P-1 were not hydrolysed. Although OP-Tc has weak arginine aminopeptidase activity, the enzyme clearly preferred substrates that had amino acids in the P-2 and P-3 positions. Overall, OP-Tc appears to be less efficient than OP-Tb because it usually displayed lower k(cat)/K-m values for the substrates tested. However, like OP-Tb, the best substrate for OP-T c was Cbz-Arg-Arg-AMC (K-m = 0.72 mu M, k(cat) = 96 s(-1)). OP-Tc preferenc e for amino acids in the P-2 position was (Gly,Lys,Arg) > Phe > Leu > Pro. The results also suggest that the P-3-binding site has hydrophobic characte ristics.