A marked gradation in active-centre properties in the cysteine proteinases revealed by neutral and anionic reactivity probes. Reactivity characteristics of the thiol groups of actinidin, ficin, papain and papaya peptidase A towards 4,4'-dipyridyl disulphide and 5,5'-dithiobis-(2-nitrobenzoate) dianion.
1. The kinetics of the reactions of the catalytic-site thiol groups of actinidin (the cysteine proteinase from Actinidia chinensis), ficin (EC 3.4.22.3), papain (EC 3.4.22.2) and papaya peptidase A (the other monothiol cysteine proteinase component of Carica papaya) with 4,4'-dipyridyl disulphide (4-Py-S-S-4-Py) and with 5,5'-dithiobis-(2-nitrobenzoate) dianion (Nbs22-) were studied in the pH range approx. 6-10. These studies provided the pH-independent second-order rate constants (k) for the reactions of the two probe reagents with the catalytic-site thiolate anions each in the environment of a neutral histidine side chain where an active-centre carboxy group would be ionized. 2. The ratio R equal to kNbs22-/k4-Py-S-S-4-Py provides an index of the catalytic-site solvation properties of the four cysteine proteinases and varies markedly from one enzyme to another, being 0.80 for papaya peptidase A (0.86 for the model thiol, 2-mercaptoethanol), 29 for actinidin, 0.18 for ficin and 0.015 for papain. These differences appear to derive mainly from the response of the enzyme to the negative charge on Nbs22-. 3. Possible implications of these results for (a) mechanisms of cysteine proteinase catalysis and (b) the possibility of using series of functionally related enzymes in the study of mechanism are discussed.
D000838:Anions / Q000378:metabolism; D001665:Binding Sites; D002917:Chymopapain / Q000378:metabolism*; D003546:Cysteine Endopeptidases; D004220:Disulfides; D004228:Dithionitrobenzoic Acid; D010450:Endopeptidases / Q000378:metabolism*; D005361:Ficain / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008623:Mercaptoethanol; D009579:Nitrobenzoates; D010206:Papain / Q000378:metabolism*; D011725:Pyridines; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds / Q000378:metabolism
D000838:Anions; D004220:Disulfides; D009579:Nitrobenzoates; D011725:Pyridines; D013438:Sulfhydryl Compounds; C007542:4,4'-dipyridyl disulfide; D008623:Mercaptoethanol; D004228:Dithionitrobenzoic Acid; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; C018745:actinidain; D010206:Papain; D005361:Ficain; D002917:Chymopapain