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.
Vignain is reported to be synthesized de novo during the initial stages of germination (Shutov & Vaintraub, 1987). But Kalinsky et al. (1992) have reported the
59 presence of P34, a vignain-like cysteine endopeptidase in maturing soybean seeds. We
have detected activity of this enzyme in the dry seeds but only after acid treatment. It has
a broad specificity and has been implicated in the mobilization of seed storage proteins
during germination. However the role of vignain in maturation of seeds is not known as
yet. Therefore, it appears that both vignain and legumain are present in the maturing as
well as germinating seeds but at present we do not know their precise physiological func
tion.
Legumain, glycylendopeptidase and clostripain, due to their ability to introduce
restricted cleavages into polypeptide substrates are valuable tools in the study of the
primary and secondary structures of proteins. A set of limit peptides which is generated by
proteolysis can be separated and sequenced. Sequence data of overlapping sets generated
with different endopeptidases can be combined to give the entire sequence. The amino
acid sequence of cathepsin D from potato (Ritonja et al., 1991) and bovine cathepsin S and
L (Ritonja et al., 1991) has been determined with the help of glycylendopeptidase as one
of the enzymes.
Since histones have large amounts of arginine, clostripain has been used for the
selective cleavage of histone H3 and H4 to obtain chromatin core particle (Encontre &
Parello, 1988).
Endopeptidases having limited specificity have been used as structural tools to
probe the conformation of soluble proteins. The endopeptidase acts on unfolded protein to
yield a defined set of peptide fragments which can be separated and subsequently
characterised. The only factor to be taken into account is the bond specificity exhibited by
the enzyme (Price & Johnson, 1989).
Recently endopeptidases have also been used as catalysts fortransacylation, i.e. the
formation of a peptide bond. Different endopeptidases are now used for the synthesis of
peptide hormones, neuropeptides, peptides sweetners (such as aspartame) and protein
modification such as semi synthesis of human insulin (Kasche, 1989).
60