E Salih

Chapter 2 Cysteine proteinases

Publisher Summary The classification of proteinases is based on their possession of analogous mechanistic devices and on their susceptibility to group specific inhibitors. The cysteine proteinases, which were previously known as thiol proteinases, constitute the group of endopeptidases whose members rely for catalytic activity on the presence of a thiol group of a cysteine residue in the enzyme molecule. The other three main classes of endopeptidase are serine proteinases, aspartic proteinases, and metalloproteinases. Although not all cysteine proteinases have been subjected to substantial mechanistic study, those that have, appear to contain a thiol–imidazole interactive system within the catalytic site and it seems probable that this could be a common feature of all enzymes in the group. A plausible component of this interactive system is a thiolate–imidazolium ion-pair, the thiolate anion of which becomes transiently acylated during catalysis, assisted by general acid catalysis provided by the imidazolium ion.

Evidence from two-protonic-state reactivity probe kinetics that chymopapain in fresh nonfruit latex ofCarica papaya consists of multiple forms of chymopapain A. The value of catalytic site characteristics in the identification, classification, and characterization of the papaya cysteine proteinases papain, the chymopapains, and papaya proteinase Ω

Fresh latex ofCarica papaya was collected from the stem, leaves, and petioles of the growing plant and fractionated by ion-exchange chromatography on a column of SP-Sephadex-C50 and by FPLC using a Mono S column. The fractions were examined for catalytic activity using Z-Lys-ONp andl-BAPNA as substrates and the thiol contents and reactivity characteristics were determined by using 2,2′-dipyridyl disulfide as a two-protonic-state thiol titrant and reactivity probe. By these methods the fresh nonfruit latex was shown to contain papain (EC 3.4.22.2), multiple forms of chymopapain, all of which have catalytic site reactivities characteristic of chymopapain A, and papaya proteinase Ω (originally called papaya peptidase A). The necessity now to characterize the catalytic site of a chymopapain in order to identify it is discussed.