The specificity of trichomonad cysteine proteinases analysed using fluorogenic substrates and specific inhibitors

The multiple cysteine proteinases of Trichomonas vaginalis and Tritrichomonas foetus, both those retained intracellularly and those released, were separated using gelatin-SDS-PAGE, and their activity towards a range of 15 fluorogenic peptidyl aminomethylcoumarins determined together with their relative sensitivity to inhibitors. Three types of enzyme were apparent in T. vaginalis: (i) an 86-kDa enzyme active only on Z-Arg-Arg-NHMec; (ii) a 54-kDa proteinase which was most active on Z-Phe-Arg-NHMec but also able to hydrolyse N-t-Boc-Val-Leu-Lys-NHMec, Suc-Ala-Phe-Lys-NHMec, H-Pro-Phe-Arg-NHMec and Z-Arg-Arg-NHMec; and (iii) a group of six enzymes which preferentially hydrolysed substrates with bulky residues at the P2 and P3 positions. N-t-Boc-Val-Leu-Lys-NHMec and H-Leu-Val-Tyr-NHMec were the best substrates for the latter group. The 86-kDa proteinase was inactivated by E-64, but only at high concentrations, and was relatively insensitive to the peptidyl diazomethanes. The other proteinases were inhibited by low concentrations of E-64 and by Z-Phe-Ala-CHN2, and to a lesser extent by Z-Phe-Phe-CHN2. Differences between the proteinases of T. foetus were also demonstrated. All of them were active on Z-Arg-Arg-NHMec, but their activity towards other substrates varied. Three predominantly extracellular proteinases (25, 27 and 34 kDa), hydrolysed Z-Arg-Arg-NHMec specifically. Other proteinases (apparent Mr of 20,000 and 32,000) hydrolysed a number of other substrates, with the 32-kDa enzyme having greater activity towards N-t-Boc-Val-Leu-Lys-NHMec and H-Leu-Val-Tyr-NHMec than towards Z-Arg-Arg-NHMec. At a high concentration (270 microM), E-64 inhibited all of the T. foetus enzymes, but lower concentrations were less effective, with the 18-kDa proteinase being particularly insensitive. Z-Phe-Ala-CHN2 and Z-Phe-Phe-CHN2 were relatively poor inhibitors. The results demonstrate that the proteinases of both species are a heterogeneous group with respect to specificity, and have highlighted significant differences between the enzymes of T. vaginalis and T. foetus. The information on the specificities will be useful for assessing the features required in proteinase inhibitors if they are to be of potential value as antitrichomonal agents.