Characterization of papaya peptidase A as a cysteine proteinase of Carica papaya L. with active-centre properties that differ from those of papain by using 2,2'-dipyridyl disulphide and 4-chloro-7-nitrobenzofurazan as reactivity probes. Use of two-protonic-state electrophiles in the identification of catalytic-site thiol groups.
1. The proteinase papaya peptidase A, one of the major components of the latex of Carica papaya L., was shown to contain 1 thiol group per molecule; this thiol group is essential for catalytic activity and is part of the catalytic site. 2. The usefulness of two-protonic-state reactivity probes coupled with modification/activity-loss data in assigning a thiol group as an integral part of the catalytic site as against merely 'essential' for activity is discussed. 3. The active centre of papaya peptidase A was investigated by using 2,2'-dipyridyl disulphide and 4-chloro-7-nitrobenzofurazan as reactivity probes. The presence in the enzyme in weakly acidic media of an interactive system containing a nucleophile S atom (pKI3.9,pKII7.9) was demonstrated. 5. Papaya peptidase A resembles ficin (EC 3.4.22.3) and actinidin (the cysteine proteinase from Actinidin chinenis) in that it does not appear to possess a carboxy group able to influence the reactivity of the thiol group by change of ionization state at pH values of about 4, a situation that contrasts markedly with that which obtains in papain. 6. Implications of the results for possible variations in cysteine proteinase mechanism are discussed.
D015082:2,2'-Dipyridyl / Q000031:analogs & derivatives / Q000494:pharmacology*; D009327:4-Chloro-7-nitrobenzofurazan / Q000494:pharmacology*; D016282:Aspartic Acid Endopeptidases; D001665:Binding Sites; D004220:Disulfides; D010450:Endopeptidases / Q000378:metabolism*; D005361:Ficain / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010069:Oxadiazoles / Q000494:pharmacology*; D010206:Papain / Q000378:metabolism; D010944:Plants / Q000201:enzymology*; D011725:Pyridines / Q000494:pharmacology*; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000494:pharmacology*
D004220:Disulfides; D010069:Oxadiazoles; D011725:Pyridines; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; C001105:2,2'-dipyridyl disulfide; D015082:2,2'-Dipyridyl; D010450:Endopeptidases; D010206:Papain; D005361:Ficain; C022325:aspartic proteinase A; D016282:Aspartic Acid Endopeptidases; D009327:4-Chloro-7-nitrobenzofurazan