The specificity of clostripain from Clostridium histolyticum. Mapping the S' subsites via acyl transfer to amino acid amides and peptides.

Abstract

The S' subsite specificity of clostripain from Clostridium histolyticum was investigated by acyl transfer to libraries of amino acid amides, Ala-Xaa dipeptides, proline derivatives and pentapeptides using N alpha-benzoyl-L-arginine ethyl ester as acyl donor. A pentapeptide library consisting of 29 pentapeptides with general structure Xaa-Ala-Ala-Ala-Gly, Ala-Xaa-Ala-Ala-Gly and Ala-Ala-Xaa-Ala-Gly, where Xaa represents Gly, Ala, Pro, Leu, Phe, Asp, Glu, Arg and Lys, was prepared by solid-phase synthesis. The data analysis was performed by HPLC and evaluated by statistical algorithms. The nucleophile efficiency covers a range of more than three orders of magnitude. In the P'1 position, low specificity for amino acid amides and Xaa-(Ala)3-Gly peptides was found, however, in the P'2 position, positively charged amino acid residues are strongly preferred. The negatively charged side chains of aspartic acid and glutamic acid in the P'1 and P'2 positions, respectively, show only poor nucleophilic behaviour. In the case of these amino acids, the S'-P' interactions depend significantly on their position of these residues in the peptide chain of the nucleophile. The transfer of aspartic acid and glutamic acid from P'1-P'3 increases the nucleophile efficiency by approximately two orders of magnitude. The aromatic side chains are not well accepted, especially in the case of P'3Phe. Surprisingly, P'1Gly leads to effective P'-S' interactions. However, the opposite result was obtained for P'2Gly. The high efficiency for Gly-NH2 does not fit with the hydrophobicity structure/activity relationships. In most cases, peptide chain elongation does not improve the nucleophile efficiency. The effective interaction of D-Leu-NH2 with the S' subsite of clostripain emphasizes the fact that the nucleophile stereospecificity is not restricted to L-amino acids. The results with proline derivatives indicate remarkably different specificities of the S' binding site which can only be explained by conformational restraints. A positive cooperativity between P'1Pro and P'2Gly and a negative cooperativity between P'1Pro and P'2Phe was observed. The arrangement of three proline residues next to each other represents a favourable conformation for effective enzyme-nucleophile interactions.