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174 RAWLINGS ET AL.
contain more than one peptidase unit in separate polypeptide chains; these we
term complex peptidases. We use a special type of identifier starting in “X”
for the compound and complex peptidases. In addition, a conventional MEROPS
identifier is assigned to each of the individual peptidase units. For example, the
somatic form of peptidyl-dipeptidase A (angiotensin-converting enzyme) is XM02-
001 (Corvol et al., 2004), and its two peptidase units are M02.001 and M02.004.
There is a summary page in the database for XM02-001 in addition to the standard
pages for M02.001 and M02.004. Other examples of compound peptidases are
meprin A complex (XM12-001; (Bertenshaw et al., 2004)) and carboxypeptidase D
(XM14-001;(Fricker, 1998)). Examples of complex peptidases are the proteasome
(XT01-001; (Seemuller et al., 2004)), AAA endopeptidase complex (XM41-001;
(Thorsness et al., 2004)), eukaryote signal peptidase (XS26-001; (Walker, 2004))
and the tricorn complex (XP01-001; (Tamura et al., 2004)). The tricorn peptidase
complex is unique in that the components belong to different peptidase families.
Peptidases that are homo-oligomers require no special classification in MEROPS
because a single identifier can encompass all the peptidase units.
3.3.6. Peptidase inhibitors
The MEROPS database also includes the protein inhibitors of peptidases (Rawlings
et al., 2004). Many inhibitors bind to the peptidase in a substrate-like way, except
that the complex is stable even if hydrolysis occurs. This mechanism is known as
the Laskowski mechanism after the scientist who characterized it (though it is
also known as the standard mechanism (Laskowski et al., 2000)). The residue
that interacts with the nucleophile of the peptidase is known as the reactive site
residue. An example of an inhibitor that uses the Laskowski mechanism is the
turkey ovomucoid third domain (I01.003).
A second mechanism is known as a trapping reaction. This kind of reaction
is specific for endopeptidases because it depends upon the cleavage of an internal
bond in the inhibitor that triggers a conformational change which either traps
the enzyme, for example in the case of alpha
2
-macroglobulin inhibition (I39.001;
(Barrett, 1981)), or disrupts the active site of the peptidase, for example alpha
1
-
peptidase inhibitor (I04.001; (Huntington et al., 2000)). Alpha
2
-macroglobulin is
able to inhibit a wide range of endopeptidases of every catalytic type because it
contains a long loop containing bonds susceptible to proteolysis known as the bait
region.
Generally, inhibitors are classified in a similar way to peptidases, and the classifi-
cation is one of inhibitor units, an inhibitor unit being that segment of the sequence
that contains a single reactive site (or bait region). There is a similar hierarchical
classification of clan, family and inhibitor.
3.3.7. Compound inhibitor
At least 12 of the families of peptidase inhibitors contain what we term compound
inhibitors; these are families I1, I2, I3, I8, I12, I15, I17, I19, I20, I25, I27 and
I31. The compound inhibitors are proteins that contain multiple inhibitor units. The
INTRODUCTION TO PEPTIDASES AND THE MEROPS DATABASE 175
number of inhibitor units ranges from 2-15 (Rawlings et al., 2004). The identifier for
each of these compound inhibitors starts with the letter “L” followed by the name
of the family to which the peptidase units belong, a hyphen, and a serial number.
For example, ovomucoid contains three inhibitor units (Kato et al., 1987). These are
I01.001, I01.002 and I01.003, and whole protein has the identifier LI01-001. The
summary page for the compound inhibitor LI01-001 contains a diagram that shows
how the individual units are arranged. A few compound inhibitors are known that
contain units from more than one family of inhibitors. These have identifiers that
start “LI90”, and an example is chelonianin (LI90-003), which contains a domain
related to I2 (I02.022) and a domain related to I17 (I17.004).
3.3.8. Type peptidase, type inhibitor
A type peptidase is nominated for each family and subfamily. All peptidases that
are homologous to the type peptidase are members of this family. Similarly, a type
inhibitor is nominated for each inhibitor family.
3.3.9. Families
The term family is used to describe a group of peptidases or peptidase inhibitors
each of which can be proved to be homologous to the type example. The homology
is shown by a significant similarity in amino acid sequence either to the type
example itself or to another protein that has been shown to be homologous to the
type example and thus a member of the family. The relationship must exist in the
peptidase unit at least. A family can contain a single peptidase if no homologues
are known, and a single gene product such as a virus polyprotein can contain more
than one peptidase each assigned to a different family.
Some families are divided into subfamilies because there is evidence of a very
ancient divergence within the family. Typically, the divergence corresponds to
more than 150 accepted point mutations per 100 amino acid residues, which would
represent an event 2,500 million years ago for a family with a typical evolutionary
rate of 0.6 substitutions per amino acid site per 1,000 million years. A putative
protein sequence that is very divergent from known peptidases in the family does
not normally found a new subfamily but is described as “unassigned” until more is
known about it.
At the time of writing, there are nearly 200 families of peptidases in MEROPS
(Release 7.2). The naming of the families follows the system introduced by Rawlings
and Barrett (Rawlings et al., 1993) in which each family is named with a letter
denoting the catalytic type (S, C, T, A, G, M or U, for serine, cysteine, threonine,
aspartic, glutamic, metallo- or unknown), followed by an arbitrarily assigned
number. For example, the caspase family of cysteine peptidases is C14. When a
family disappears, usually because it is merged with another, the family name is
not re-used. For this reason, there are interruptions in the numerical sequences of
families that are of no current significance.
MEROPS (Release 7.2) contains 52 families of peptidase inhibitors. Because a
number of families contain inhibitors of peptidases of more than one catalytic type,
176 RAWLINGS ET AL.
it is not feasible to name the families of inhibitors according to catalytic types of the
peptidases inhibited. Consequently a single series of inhibitor family names is used,
formed from the letter “I” followed by a serial number. For example, family I4
(the “serpins”) contains serine peptidase inhibitors such as alpha
1
-antichymotrypsin
(I04.002), but also the viral serpin CrmA (I04.028), which additionally inhibits the
cysteine peptidase caspase 1 (Komiyama et al., 1994).
3.3.10. Clans
In a clan we include all the modern-day peptidases that we believe to have arisen
from a single evolutionary origin of peptidases, although they commonly have
diverged so far that they now belong in more than one family. The homology of
peptidases in different families in a clan is most clearly shown by their similar
protein folds. The significance of the similarity can often be quantified by use
of the DALI program (Holm et al., 1997). When structures are not available, the
order of catalytic-site residues in the polypeptide chain and sequence motifs around
them may provide less direct evidence of homology at the clan level. Each clan
is identified with two letters the first of which represents the catalytic type of the
families included in the clan. The letter “P” is used for a clan containing families of
more than one of the catalytic types serine, threonine and cysteine. Some families
cannot yet be assigned to any clan, and when a formal assignment is required, such
a family is described as belonging to clan A-, C-, M-, S-, T- or U-, according to the
catalytic type. Some clans are divided into subclans because there is evidence of
a very ancient divergence within the clan. Clan MA contains subclan MA(E), the
gluzincins, and subclan MA(M), the metzincins. Clan PA is divided into subclan
PA(S), containing families of serine peptidases, and subclan PA(C), containing
families of cysteine peptidases. About 50 clans of peptidases are recognised in
MEROPS (Release 7.2).
The families of proteins that inhibit peptidases are assigned to clans in similar
ways to the families of peptidases. MEROPS (Release 7.2) contains 32 clans of
inhibitors. Identifiers are taken from the ranges IA-IZ and JA-JZ.
3.3.11. Strengths of the MEROPS classification system
Peptidases and their inhibitors represent a hot-spot of scientific research on which
thousands of scientists are working worldwide in academia and industry. The
MEROPS database provides the community with a comprehensive, integrated
resource. The hierarchical system of classification of peptidases and inhibitors
that MEROPS provides is now accepted generally as authoritative. The MEROPS
system allows for the efficient storage and retrieval of information – both within
the database itself and beyond.
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CHAPTER 11
CYSTEINE PROTEASES
ZBIGNIEW GRZONKA, FRANCISZEK KASPRZYKOWSKI
AND WIESŁAW WICZK
∗
Faculty of Chemistry, University of Gda
´
nsk, Poland
∗
ww@chem.univ.gda.pl
1. INTRODUCTION
Cysteine proteases (CPs) are present in all living organisms. More than twenty
families of cysteine proteases have been described (Barrett, 1994) many of
which (e.g. papain, bromelain, ficain , animal cathepsins) are of industrial impor-
tance. Recently, cysteine proteases, in particular lysosomal cathepsins, have
attracted the interest of the pharmaceutical industry (Leung-Toung et al., 2002).
Cathepsins are promising drug targets for many diseases such as osteoporosis,
rheumatoid arthritis, arteriosclerosis, cancer, and inflammatory and autoimmune
diseases. Caspases, another group of CPs, are important elements of the apoptotic
machinery that regulates programmed cell death (Denault and Salvesen, 2002).
Comprehensive information on CPs can be found in many excellent books and
reviews (Barrett et al., 1998; Bordusa, 2002; Drauz and Waldmann, 2002; Lecaille
et al., 2002; McGrath, 1999; Otto and Schirmeister, 1997).
2. STRUCTURE AND FUNCTION
2.1. Classification and Evolution
Cysteine proteases (EC.3.4.22) are proteins of molecular mass about 21-30 kDa.
They catalyse the hydrolysis of peptide, amide, ester, thiol ester and thiono ester
bonds. The CP family can be subdivided into exopeptidases (e.g. cathepsin X,
carboxypeptidase B) and endopeptidases (papain, bromelain, ficain, cathepsins).
Exopeptidases cleave the peptide bond proximal to the amino or carboxy termini of
the substrate, whereas endopeptidases cleave peptide bonds distant from the N- or
C-termini. Cysteine proteases are divided into five clans: CA (papain-like enzymes),
181
J. Polaina and A.P. MacCabe (eds.), Industrial Enzymes, 181–195.
© 2007 Springer.
182 GRZONKA ET AL.
CB (viral chymotrypsin-like CPs), CC (papain-like endopeptidases of RNA viruses),
CD (legumain-type caspases) and CE (containing His, Glu/Asp, Gln, Cys residues in
the catalytic cleft) (Barrett, 1994, 1998; Rawlings et al., this volume). The majority
of CPs that have been characterized are evolutionarily related to papain and share
a common fold. They are synthesized as inactive precursors with a N-terminal
propeptide and a signal peptide. Some peptidases of family C1 have C-terminal
extensions. Activation requires proteolytic cleavage of the N-terminal proregion
that also functions as an inhibitor of the enzyme. Most CPs are inhibited by E-64,
cystatins and many synthetic inhibitors (Otto and Schirmeister, 1997; Grzonka
et al., 2001).
2.2. Papain
Papain (EC 3.4.22.2) is the best known cysteine protease. It was isolated in 1879 from
the fruits of Carica papaya and was also the first protease for which a crystallographic
structure was determined (Drenth et al., 1968; Kamphuis et al., 1984). The crude dried
latex of papaya fruit contains a mixture of at least four cysteine proteases (papain,
chymopapain, caricain, glycyl endopeptidase) and other enzymes (Baines and Brock-
lehurst,1979).Crudepapainofthehighestquality andactivity isfound insunny regions
of constant humidity throughout the year. Methods of purification of papain include
water extraction with reducing and chelating agents, salt precipitation and solvent
extraction. Very pure papain is obtained by affinity chromatography methods. Papain
is composed of 212 amino acids with three internal disulphide bridges, resulting in a
molecular weight of 23.4 kDa. It is relatively basic protein, with a pI of 8.75. Its three-
dimensional structure reveals that the enzyme is composed of two domains of similar
size with the active cleft located between them (Fig. 1).
The general mechanism of cysteine protease action has been very well studied,
with papain as the model enzyme. The enzymatic activity of papain is exerted by
a catalytic dyad formed by Cys
25
and His
159
residues, which in the pH interval
3.5-8.0 form an ion-pair (Fig. 2). Asn
175
is important for orientation of the imida-
zolium ring of the histidine in the catalytic cleft. The reactive thiol group of the
enzyme has to be in the reduced form for catalytic activity. Thus, the cysteine
proteases require a rather reducing and acidic environment to be active. The
formation of an intermediate, S-acyl enzyme moiety, is a fundamental step in
hydrolysis. This intermediate is formed via nucleophilic attack of the thiolate group
of the cysteine residue on the carbonyl group of the hydrolysed amide (ester) bond
with the release of the C-terminal fragment of the cleaved product. In the next
step, a water molecule reacts with the intermediate, the N-terminal fragment is
released, and the regenerated free CP molecule can begin a new catalytic cycle
(Storer and Menard, 1994).
The active site residues Cys
25
and His
159
are positioned on opposite sides of
the cleft. A number of structures of papain complexes with ligands and inhibitors
have been elucidated by X-ray crystallography. Following the notation of Schechter
and Berger (1967), the substrate pocket of papain binds at least seven amino acid
CYSTEINE PROTEASES 183
Figure 1. Ribbon representation of the three-dimensional structure of papain (Kamphuis et al., 1984)
Figure 2. Enzymatic mechanism of protein hydrolysis by cysteine proteases
184 GRZONKA ET AL.
residues in appropriate S
n
and S
n
’ subsites (Fig. 3). On the basis of kinetic and
structural data Turk et al. (1998) proposed that only five subsites are important
for substrate binding. According to their proposal, the S
2
,S
1
and S
1
’ subsites
are important for both backbone and side-chain binding, whereas the S
3
and S
2
’
pockets are crucial only for amino acid side-chain binding. A preference for those
substrates containing a bulky hydrophobic chain (Phe, Leu, Ile etc.) in P
2
position
was found; the amino acid residue in position P
1
of the substrate influences substrate
binding to the enzyme to a lesser degree. There is some preference for basic amino
acids (Arg, Lys) in this position but Val is not accepted. The S
3
binding site of the
enzyme is less constrained; it can accommodate different amino acids side chains.
Generally, papain possesses fairly broad specificity and can cleave various peptide
bonds. The optimal activity of papain occurs at pH 5.8–7.0 and at temperature
50–57
C when casein is used as the substrate. Papain is stable and active for several
months when stored at 4
C. Decreased activity during storage is due to oxidation of
the active site thiol group. This oxidation can be partially reversed by thiol reagents
(cysteine, mercaptoethanol, dimercaptopropanol etc.).
2.3. Bromelain
The name ‘bromelain’ was originally given to the mixture of proteases found in the
juice of the stem and fruit of pineapple (Ananas comosus). Even now, bromelain
is still used as the collective name for enzymes found in various members of
the Bromeliaceae family. The major endopeptidase present in extracts of plant
stem is termed ‘stem bromelain’, whereas the major enzyme fraction found in the
juice of the pineapple fruit is named ‘fruit bromelain’. Some other minor cysteine
endopeptidases (ananain, comosain) are also found in the pineapple stem.
Stem bromelain (EC 3.4.22.32) belongs to the papain family. It is a glycosylated
single-chain protein of molecular weight 24.5 kDa. It contains 212 amino acid
residues, including seven cysteines, one of which is involved in catalysis. The other
six are associated in pairs forming three disulphide bridges. The crystal structure
of stem bromelain has not yet been reported. Stem bromelain can be purified
Figure 3. Interaction of papain with substrate