Subsite specificity of trypanosomal cathepsin L-like cysteine proteases - Probing the S2 pocket with phenylalanine-derived amino acids

The S2 subsite of mammalian cysteine proteinases of the papain family is es sential for specificity. Among natural amino acids, all these enzymes prefe r bulky hydrophobic residues such as phenylalanine at P2. This holds true f or their trypanosomal counterparts: cruzain from Trypanosoma cruzi and cong opain from T. congolense. A detailed analysis of the S2 specificity of para sitic proteases was performed to gain information that might be of interest for the design of more selective pseudopeptidyl inhibitors. Nonproteogenic phenylalanyl analogs (Xaa) have been introduced into position P2 of fluoro genic substrates dansyl-Xaa-Arg-Ala-Pro-Trp, and their kinetic constants (K -m, k(cat)/K-m) have been determined with congopain and cruzain, and relate d host cathepsins B and L. Trypanosomal cysteine proteases are poorly stere oselective towards d/l-Phe, the inversion of chirality modifying the effici ency of the reaction but not the K-m. Congopain binds cyclohexylalanine bet ter than aromatic Phe derivatives. Another characteristic feature of congop ain compared to cruzain and cathepsins B and L was that it could accomodate a phenylglycyl residue (k(cat)/K-m = 1300 mm(-1).s(-1)), while lengthening of the side chain by a methylene group only slightly impaired the specific ity constant towards trypanosomal cysteine proteases. Mono- and di-halogena tion or nitration of Phe did not affect K-m for cathepsin L-like enzymes, b ut the presence of constrained Phe derivatives prevented a correct fitting into the S2 subsite. A model of congopain has been built to study the fit o f Phe analogs within the S2 pocket. Phe analogs adopted a positioning withi n the S2 pocket similar to that of the Tyr of the cruzain/Z-Tyr-Ala-fluorom ethylketone complex. However, cyclohexylalanine has an energetically favora ble chair-like conformation and can penetrate deeper into the subsite. Fitt ing of modeled Phe analogs were in good agreement with kinetic parameters. Furthermore, a linear relationship could be established with logP, supporti ng the suggestion that fitting into the S2 pocket of trypanosomal cysteine proteases depends on the hydrophobicity of Phe analogs.