CHAPTER V General Discussion GENERAL DISCUSSION The cysteine endopeptidases represent one of the four classes of enzymes that act on peptide bonds of proteins and oligopeptides. Papain, the protogonist of the cysteine endopeptidases has been by far the most extensively studied of this class of enzymes. Most of the cysteine endopeptidases characterised so far show a high degree of similarity with regard to their physico-chemical properties, specificity and primary and secondary structures to papain, and are now recognised as papain superfamily. Rawlings and Barrett, (1993) have classified endopeptidases into different families based on the sequence homology and active site residues. They showed that there are 14 different families of cysteine endopeptidases and the papain family is the largest. In the absence of sequence data, kinetic parameters of inhibitor binding and knowledge of active site residues provides ample scope to classify endopeptidases at least into papain and nonpapain families. Hence, based on these information an attempt was made to classify the cysteine endopeptidases investigated in these studies. Vignain, legumain, glycylendopeptidase (papaya proteinase IV) and clostripain are activated in the presence of thiols and have a pH optimum of 5-7, a general characteristics for enzymes belonging to the cysteine class. The molecular weight of enzymes belonging to the papain family fall in the range of 23 kDa - 28 kDa with papain having a molecular weight of 23.35 kDa. Vignain (28 kDa) and glycylendopeptidase (25 kDa) have molecular weights in this range, whereas clostripain and legumain have a molecular weights of 58 kDa and 33 kDa, respectively, which are higher than that of papain. The isoelectric point of papain is around pH 8.75 and only glycylendopeptiadse has a basic pi. Vignain, legumain and clostripain are acidic proteins and their pis are in the range of pH 4 - 5.5. Substrate specificity: Using synthetic substrates and proteins of known sequences such as B chain of oxidised insulin, the specificity of endopeptidases for a particular bond can be evaluated. Such studies have been done for papain and their homologues. Papain is known to have a strong preference for Phe, Tyr, Val or Leu at the P2-position of a 56 substrate. In general, a preference for the bulky hydrophobic group at P2 has been report­ ed for ficin (Kortt et ai, 1974), bromelain (Wharton, 1974), streptococcal proteinase (Liu et ai, 1969) and cathepsin B (Green and Shaw, 1981) and L (Towatari and Katunuma, 1983). Vignain has a papain like specificity with a preference for arginine in the Pj position. But the specificities of legumain, glycylendopeptidase and clostripain are highly restricted. Legumain cleaves only asparagine bonds, unlike clostripain and glycylendopeptidase which cleave arginine and glycine bonds respectively. These enzymes have unique specificity although they catalyse the same reaction i.e. cleaving of the peptide bond. The amino-acid sequence and the x-ray crystallographic studies probably would provide the details of structural features responsible for imparting these unique specificities. In the absence of such an information, we have tried to understand the environment in the active site of these enzymes with the help of kinetic parameters of substrate and inhibitor binding. Inhibitor specificity: Members of the papain family have some distinguishing properties as endopeptidases. The enzymes belonging to this family were found to be highly susceptible to E-64 (the epoxide inhibitor), cystatin (the protein inhibitor of cysteine endopeptidases), and haloacetate and haloacetamide. E-64 reacts rapidly with cysteine endopeptidases of the papain superfamily (Table V.l). The second order rate constant with papain and cathepsin B was 638,000 M"1is" 11 and 89,000 M'V1 respectively (Barrett et ai, 1982) (Table V.2). In comparison vignain had a rate of 34,965 M . s"1 whereas glycylendopeptidase reacted much more slowly with a rate of 15,300 M . s . There was no inhibition of legumain even at a final concentration of 2 mM and only a reversible type of inhibition was seen with clostripain with Ki values greater than 20 nM. The reversible inhibition of clostripain could be due to E-64 binding in a non-productive way and the agmatine of the E-64 being cleaved to make most of the molecule noninhibitory. Cystatin is a tight binding inhibitor of papain and its homologues, with a ki value of 0.005 nM for papain. Vignain binds to cystatin with a Ki of 30 \iM. In comparison, 57 Table V.l : Inhibitions of cysteine endopeptidases by E-64 Inhibited Not inhibited Papain Legumain Cathepsin B Clostripain Cathepsin L (reversible inhibition) Ficin Bromelain Glycyl endopeptidase Vignain Table V.2 : Comparison of A'2 values for inhibition of cysteine endopeptidase by E-64 Enzyme A'2 M 1,-s 1 Papain 638,000 Cathepsin B 89,400 Cathepsin L 96,250 Glycyl endopeptidase 15,300 Vignain 34,965 _ Clostripain no inhibition Legumain no inhibition glycylendopeptidase, legumain and clostripain were bound extremly loosely with several orders of magnitude difference in the Ki values. (Table V.3). Other cysteine endopeptidases showing very weak binding to cystatin are fruit and stem bromelain (Buttle etai, 1990). The rate of interaction of the active thiol and the imidazole group with iodoacetate differs with different cysteine endopeptidases. However, the enzymes belonging to papain superfamily exhibit a typical behavior of a very fast reaction with iodoacetate and comparatively slower reaction with neutral iodoacetamide. Polgar and Halasz, (1977) compared the rate of inactivation of papain and thiolsubtilisin (where the active serine is replaced by cysteine) with iodoacetate and iodoacetamide. They found that iodoacetic acid reacted much faster with papain and the ratio of the two rate constants was 0.05. The artificial enzyme, thiolsubtilisin had a ratio of 7.5, indicating that the reaction with iodoacetamide was much faster than iodoacetate. Based on this data it was concluded that the difference in the geometries in the ion-pairs is responsible for the basic difference in the catalytic abilities of papain and thiolsubtilisin. Halasz and Polgar (1977) stated that this difference in the rates cannot be due to steric hindrance by some amino acid residues since it is obvious from the three-dimensional model of the active sites that the small alkylating agent can readily approach the reacting sulphur atom in both the enzymes. It is likely that the polarity of the microenvironment of their active site is different and the sulphur atom of thiolsubtilisin is located in a relatively nonpolar environment. Hence there could be difficulty in transferring a negative charge in this environment. By carrying out similar studies with vignain, legumain, glycylendopeptidase and clostripain, it was observed that all the four enzymes had much slower rates for iodoacetate and iodoacetamide compared to papain (Table V.4). Vignain and glycylendopeptidase reacted faster with iodoacetate than iodoacetamide. On the other hand legumain and clostripain reacted much more slowly with iodoacetamide than iodoacetate. It is evident that of the four enzymes studied, legumain and clostripain are non 58 Table V.3 : Comparison of K{ values for inhibition of cysteine endopeptidase with cystatin Enzyme K% (nm) Papain 0.005 Cathepsin B 0.81 Cathepsin L 0.02 Ficin 0.00005 Actinidin 5.0 . Chymopapain 0.33 Vignain 30 Legumain >> 5000 Clostripain > 500 Glycyl endopeptidase >> 1130 Fruit bromelain >> 1100 Stem bromelain > 36000 Table V.4 : &2 value for alkylation of ion pair by iodoacetate and iodoacetamide IAA IAN IAN/IAA M_1s_1 M~ls~l ratio Papain 1100 14.5 0.05 Stem bromelain 17.7 5.5 0.31 Vignain 1.56 0.270 0.173 Glycylendopeptidase 2.2 0.5 0.23 Thiol subtilisin 0.84 6.3 7.5 Legumain 0.204 2.3 11.27 (Vigna aconitifolia ) Legumain 0.1 20.1 181.82 (Phaseolus vulgaris) Clostripain 0.79 5.2 6.58 papain-iike and glycylendopeptidase and vignain are papain-like. This conclusion is drawn from the fact that legumain and clostripain are not inhibited by E-64 and their rate constant ratios for iodoacetamide and iodoacetic acid are unlike that of papain. Although glycylendopeptidase has got a unique specificity and binds weakly to cystatin, its inhibition with E-64 and iodoacetamide/iodoacetic acid ratios are comparable to papain. Possible physiological role and application The existence of legumain like endopeptidase, having limited specificity for asparagine bonds has been reported in dry and maturing seeds (Scott et al., 1992; Bowles et al, 1986) as well as germinating seeds (Csoma and Polgar, 1984). It appears that this enzyme could have a role in the maturation of seeds, where post-translational modification of proteins takes place by limited proteolysis before being deposited in the protein bodies, as in the case of concanavalin A (Carrington et al., 1986). In soybean, it has been reported that the post-translational processing of pro-P34 (a cysteine endopeptidase) to mature P34, apparently involves the cleavage on the carboxyl side of asparagine 122 (Kalinsky et al., 1992). Asparagine specific processing has also been demonstrated in a wide variety of plant seed vacuolar proteins, such as the US superfamily of storage proteins. During germination, the involvement of legumain-like enzymes in the initial proteolytic cleavage of the storage proteins in the cotyledons has been suggested (Boylan & Sussex, 1987). The seed storage proteins may be stored in a form which makes it inaccessible to general proteolysis. The "nicking" action of the specific endopeptidase may be needed to make the protein available for less specific endopeptidases which then complete the digestion. According to Shutov and Vaintraub (1987) the more general endopeptidase do the nicking action followed by complete digestion with legumain-like endopeptidase. Therefore, legumain could have different biological roles at different times during maturation and germination of legume seeds.