1. The selectivity observed when the potentially general technique for the isolation of fully active forms of cysteine proteinases, covalent chromatography by thiol-disulphide interchange, is applied to chymopapain M and to actinidin was investigated by a combination of experimentation and computer modelling. Neither of these enzymes is able to react with the original Sepharose-GSH-2-dipyridyl disulphide gel, but fully active forms of both enzymes are obtained by using Sepharose-2-hydroxypropyl-2′-dipyridyl disulphide gel, which is both electrically neutral and sterically less demanding than the GSH gel. Electrostatic potential calculations, minimization and molecular-dynamics simulations provide explanations for the unusual, but different, specificities exhibited by actinidin and chymopapain M in the interactions of their active centres with ligands. 2. The unique behaviour of chymopapain M in exerting an almost absolute specificity for substrates with glycine at the P1 position and in resisting inhibition by cystatin was examined by the computer-modelling techniques. A new, modelled, structure of the complete chicken egg-white cystatin molecule based on the crystal structure of a short form of cystatin was deduced as a necessary prerequisite. The results suggest that electrostatic repulsion prevents reaction of actinidin with the GSH gel, whereas a steric ‘cap’ resulting from a unique arginine-65-glutamic acid-23 interaction in chymopapain M prevents reaction of the gel with this enzyme and accounts for the lack of its inhibition by cystatin and its specificity in catalysis. 3. Use of chymopapain M as a structural variant of papain demonstrates the validity of the predictions of Lowe and Yuthavong [Biochem. J. (1971) 124, 107-115] relating to the structural requirements and binding characteristics of the S1 subsite of papain.
The structural origins of the unusual specificities observed in the isolation of chymopapain M and actinidin by covalent chromatography and the lack of inhibition of chymopapain M by cystatin
M P Thomas, C Verma, S M Boyd, K Brocklehurst; The structural origins of the unusual specificities observed in the isolation of chymopapain M and actinidin by covalent chromatography and the lack of inhibition of chymopapain M by cystatin. Biochem J 15 February 1995; 306 (1): 39–46. doi: https://doi.org/10.1042/bj3060039
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