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.
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.
Thomas
M P
MP
Department of Biochemistry, Queen Mary and Westfield College, University of London, U.K.
Verma
C
C
Boyd
S M
SM
Brocklehurst
K
K
D001665:Binding Sites; D002845:Chromatography; D002917:Chymopapain / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D003198:Computer Simulation; D015891:Cystatins / Q000494:pharmacology*; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D004563:Electrochemistry; D005638:Fruit / Q000201:enzymology; D005978:Glutathione / Q000378:metabolism; D008958:Models, Molecular; D015394:Molecular Structure; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D015891:Cystatins; D004220:Disulfides; D003546:Cysteine Endopeptidases; C018745:actinidain; D002917:Chymopapain; D005978:Glutathione