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 July 1982
- journal article
- research article
- Published by Portland Press Ltd. in Biochemical Journal
- Vol. 205 (1) , 205-211
- https://doi.org/10.1042/bj2050205
Abstract
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.This publication has 16 references indexed in Scilit:
- [24] Two-protonic-state electrophiles as probes of enzyme mechanismPublished by Elsevier ,1982
- Evidence that the lack of high catalytic activity of thiolsubtilisin towards specific substrates may be due to an inappropriately located proton-distribution system. Demonstration of highly nucleophilic character of the thiol group of thiolsubtilisin in the catalytically relevant ionization state of the active centre by use of a two-protonic-state reactivity probeBiochemical Journal, 1981
- Evidence for a two-state transition in papain that may have no close analogue in ficin. Differences in the disposition of cationic sites and hydrophobic binding areas in the active centres of papain and ficinBiochemical Journal, 1980
- Specific covalent modification of thiols: Applications in the study of enzymes and other biomoleculesInternational Journal of Biochemistry, 1979
- Evidence that binding to the S2-subsite of papain may be coupled with catalytically relevant structural change involving the cysteine-25–histidine-159 diad. Kinetics of the reaction of papain with a two-protonic-state reactivity probe containing a hydrophobic side chainBiochemical Journal, 1979
- Isolation and characterization of papaya peptidase A from commercial chymopapainBiochemistry, 1975
- Reactions of papain and of low-molecular-weight thiols with some aromatic disulphides. 2,2′-Dipyridyl disulphide as a convenient active-site titrant for papain even in the presence of other thiolsBiochemical Journal, 1973
- Shapes of curves of pH-dependence of reactionsBiochemical Journal, 1973
- The mutability of stem bromelain: evidence for perturbation by structural transitions of the parameters that characterize the reaction of the essential thiol group of bromelain with 2,2′-dipyridyl disulphideBiochemical Journal, 1972
- A STUDY OF SOME THIOL ESTER HYDROLYSES AS MODELS FOR THE DEACYLATION STEP OF PAPAIN-CATALYSED HYDROLYSESBiochemical Journal, 1965