Assignment

On

Protease inhibitors

Submitted to:
Dr. Sashi Madaan
Dapartment of Biochemistry

Submitted by:
Punesh
Ph.D. Biochemistry
INTRODUCTION

Protein turnover is an important process in living systems. Proteins that have

served their purpose must be degraded so that their constituent amino acids can be
recycled for the synthesis of new proteins. Proteins ingested in the diet must be broken
down into small peptides and amino acids for absorption in the gut. Furthermore,
proteolytic reactions are important in regulating the activity of certain enzymes and other
proteins.
Proteases cleave proteins by a hydrolysis reaction the addition of a molecule of
water to a peptide bond:

Although the hydrolysis of peptide bonds is thermodynamically favored, such hydrolysis

reactions are extremely slow. In the absence of a catalyst, the half-life for the hydrolysis
of a typical peptide at neutral pH is estimated to be between 10 and 1000 years. Yet,
peptide bonds must be hydrolyzed within milliseconds in some biochemical processes.
The inhibitors are proteins that interact with proteases and suppress their
proteolytic activity. Such inhibitors are widespread in plant tissues and specific for one of
the four types of proteases (serine, cysteine, and aspartyl proteases and metalloproteases).
We focus on inhibitors of serine and cysteine proteases, because these inhibitors prevail
in plants and, on the other hand, their target enzymes are most common in insects.
Protein inhibitors of proteinases are widely distributed in higher plants. These
enzymes were mostly isolated from leguminous seeds. Soybean inhibitors were the most
thoroughly examined with respect to their structures and mechanisms of interaction with
proteases. Various inhibitors were isolated from cereal seeds (wheat, rice, and maize).
The great interest in studies of proteinase inhibitors from seeds is primarily related to
their possible involvement in the regulation of protein synthesis and degradation, as well
as in plant defenses against insects and microorganisms. In addition, proteinase inhibitors
attract the attention of researchers as substances capable of influencing the nutritional
value of proteins for humans and animals.
Serine and cysteine protease inhibitors are mostly small proteins, 6–25 kDa in
molecular weight. Their content in seeds reaches 4–10% of the total water-soluble
protein. The protective role of such inhibitors is supported by their preferential
association with seeds, which are most attractive to various pests and most vulnerable
because of their metabolic inactivity and the consequent lack of an active defense system.
Moreover, inhibitors are highly stable under unfavorable conditions (extreme pH, higher
temperatures, etc.). Another argument in favor of the protective role of protease inhibitors
is that inhibitors originating from the same source greatly vary in specificity. It is
noteworthy that most inhibitors exert no effect on cognate seed proteases and highly
efficiently inhibit animal and bacterial proteolytic enzymes as well as proteases of the
digestive tract of insect pests. An important field of research is seeking for new inhibitors
specific for major digestive proteases of insects. Studies of the Colorado beetle digestive
complex showed that aspartyl protease starts hydrolysis of the protein component of the
food. Hence, expression of a gene for its inhibitor in potato is potentially more effective
than expression of the oryzacystatin gene and may play a crucial role in suppressing
insect digestion

Interactions of proteases with inhibitors:

Proteolytic activation of zymogens is an irreversible process. This creates a need
for additional mechanisms regulating post-translationally activated proteases. Modulation
by specific inhibitors is the most common of these. However, in bacteria, endogenous
proteins inhibiting the proteases are relatively rare. Higher organisms have developed
various inhibitors of proteolytic enzymes, not only to control self-produced proteases.
Protease inhibitors are also an important element of the host anti-pathogen defence
system. Mammal defence protease inhibitors belong to two classes: the active-site
inhibitors, represented by a superfamilies of serpins and cystatins; and the α2-
macroglobulins. The members of the former group inactivate enzymes by binding to the
active site, the latter act as molecular traps for the proteases. However, some bacteria
have learned to utilize the inhibitors to regulate the activity of their extracellular
proteases for their own purposes.

Protease Inhibitors Are Important Drugs:

Compounds that block or modulate the activities of proteases can have dramatic
biological effects. Most natural protease inhibitors are similar in structure to the peptide
substrates of the enzyme that each inhibits. Several important drugs are protease
inhibitors. For example, captopril, an inhibitor of the metalloprotease angiotensin-
converting enzyme (ACE), has been used to regulate blood pressure. Crixivan, an
inhibitor of the HIV protease, is used in the treatment of AIDS. This protease cleaves
multidomain viral proteins into their active forms; blocking this process completely
prevents the virus from being infectious. To prevent unwanted side effects, protease
inhibitors used as drugs must be specific for one enzyme without inhibiting other proteins
within the body.
Crixivan is constructed around an alcohol that mimics the tetrahedral
intermediate; other groups are present to bind into the S2, S1, S1 , and S2 recognition
sites on the enzyme. The results of x-ray crystallographic studies revealed the structure of
the enzyme-Crixivan complex, showing that Crixivan adopts a conformation that
approximates the twofold symmetry of the enzyme. The active site of HIV protease is
covered by two apparently flexible flaps that fold down on top of the bound inhibitor.
The hydroxyl group of the central alcohol interacts with two aspartate residues of the
active site, one in each subunit. In addition, two carbonyl groups of the inhibitor are
hydrogen bonded to a water molecule (not shown), which, in turn, is hydrogen bonded to
a peptide NH group in each of the flaps. This interaction of the inhibitor with water and
the enzyme is not possible with cellular aspartyl proteases such as renin and thus may
contribute to the specificity of Crixivan and other inhibitors for HIV protease.

Specific Inhibitors of Some Proteolytic Enzymes:

The conversion of a zymogen into a protease by cleavage of a single peptide bond
is a precise means of switching on enzymatic activity. However, this activation step is
irreversible, and so a different mechanism is needed to stop proteolysis. Specific protease
inhibitors accomplish this task. For example, pancreatic trypsin inhibitor, a 6-kd
protein, inhibits trypsin by binding very tightly to its active site. The dissociation constant
of the complex is 0.1 pM, which corresponds to a standard free energy of binding of
about -18 kcal mol-1 (-75 kJ mol-1). In contrast with nearly all known protein assemblies,
this complex is not dissociated into its constituent chains by treatment with denaturing
agents such as 8 M urea or 6 M guanidine hydrochloride.
The reason for the exceptional stability of the complex is that pancreatic trypsin
inhibitor is a very effective substrate analog. X-ray analyses showed that the inhibitor lies
in the active site of the enzyme, positioned such that the side chain of lysine 15 of this
inhibitor interacts with the aspartate side chain in the specificity pocket of trypsin. In
addition, there are many hydrogen bonds between the main chain of trypsin and that of its
inhibitor. Furthermore, the carbonyl group of lysine 15 and the surrounding atoms of the
inhibitor fit snugly in the active site of the enzyme. Comparison of the structure of the
inhibitor bound to the enzyme with that of the free inhibitor reveals that the structure is
essentially unchanged on binding to the enzyme. Thus, the inhibitor is preorganized into
a structure that is highly complementary to the enzyme's active site. Indeed, the peptide
bond between lysine 15 and alanine 16 in pancreatic trypsin inhibitor is cleaved but at a
very slow rate: the half-life of the trypsin-inhibitor complex is several months. In
essence, the inhibitor is a substrate, but its intrinsic structure is so nicely complementary
to the enzyme's active site that it binds very tightly and is turned over slowly.
Why does trypsin inhibitor exist? Indeed, the amount of trypsin is much greater
that that of the inhibitor. Under what circumstances is it beneficial to inhibit trypsin?
Consequently, it is vital that even small amounts of trypsin be prevented from initiating
the cascade prematurely. Trypsin molecules activated in the pancreas or pancreatic ducts
could severely damage those tissues, leading to acute pancreatitis. Tissue necrosis may
result from the activation of proteolytic enzymes (as well as prolipases) by trypsin, and
hemorrhaging may result from its activation of elastase. We see here the physiological
need for the tight binding of the inhibitor to trypsin.
Pancreatic trypsin inhibitor is not the only important protease inhibitor. a 1
-Antitrypsin (also called a 1 -antiproteinase), a 53-kd plasma protein, protects tissues
from digestion by elastase, a secretory product of neutrophils (white blood cells that
engulf bacteria). Antielastase would be a more accurate name for this inhibitor, because it
blocks elastase much more effectively than it blocks trypsin. Like pancreatic trypsin
inhibitor, a 1-antitrypsin blocks the action of target enzymes by binding nearly
irreversibly to their active sites. Genetic disorders leading to a deficiency of a 1-
antitrypsin show that this inhibitor is physiologically important. For example, the
substitution of lysine for glutamate at residue 53 in the type Z mutant slows the secretion
of this inhibitor from liver cells. Serum levels of the inhibitor are about 15% of normal in
people homozygous for this defect. The consequence is that excess elastase destroys
alveolar walls in the lungs by digesting elastic fibers and other connective-tissue proteins.
The resulting clinical condition is called emphysema (also known as destructive lung
disease). People with emphysema must breathe much harder than normal people to
exchange the same volume of air, because their alveoli are much less resilient than
normal. Cigarette smoking markedly increases the likelihood that even a type Z
heterozygote will develop emphysema. The reason is that smoke oxidizes methionine 358
of the inhibitor, a residue essential for binding elastase. Indeed, this methionine side
chain is the bait that selectively traps elastase. The methionine sulfoxide oxidation
product, in contrast, does not lure elastase, a striking consequence of the insertion of just
one oxygen atom into a protein. There is another protease inhibitor, antithrombin III,
which is involved in the control of blood clotting.

Plant Protease inhibitors:

Many authors pointed out that protein inhibitors of proteolytic enzymes are
important factors of plant defense, primarily against insects and phytopathogenic
microorganisms. Despite certain common features, responses of plants to injury inflicted
by insects and pathogenic microorganisms are different. These differences are found in
the mechanisms of signal transduction and certain features of responses. Thus, it is
expedient to consider separately the involvement of inhibitors of proteolytic enzymes in
plant defenses against insects and pathogenic microorganisms.
The first data suggesting that inhibitors of proteolytic enzymes are involved in
plant defenses against insects were obtained in the mid-1960s: proteins acting as specific
inhibitors of intestinal proteolytic enzymes of insects of the genus Tribolium, which
cause severe losses of grain and grain processing products, were isolated from soy and
wheat seeds ( Triticum aestivum L.). Another important line of evidence comes from
experiments with tomato and potato leaves; their mechanical injury or damage done by
Colorado potato beetle (Leptinotarsa decemlineata Say.) caused systemic induction of
serine protease inhibitors. Systemic induction of proteinase inhibitors by plant tissue
injury was most extensively studied in experiments with representatives of the family
Solanacea, tomatoes, potato (Solanum tuberosum L.), and tobacco.

Plant protease inhibitors family:

1. Family of the Baumann–Birk inhibitor: A soybean protein distinct from SKTI was first
described by Baumann in 1946 and characterized in detail by Birk et al.. A molecule of
this protein, comprising 71 amino acid residues, contains seven disulfide bonds and has a
molecular weight of 8 kDa. The ability to bind trypsin and chymotrypsin at the same
time, with the formation of ternary complexes, is a characteristic feature of this inhibitor.
The molecule of the inhibitorn is composed of two homologous domains.

2. Serpins: Serpins constitute a unique family of serine proteinase inhibitors, the

members of which are found in plants, animals, and viruses. The mechanism whereby
serpins interact with the enzymes differs from the classic interaction characteristic of all
other serine proteinase inhibitors of plant origin. Specifically, it involves cleavage of the
peptide bond P1–P1' in the molecule of the inhibitor and the formation of a covalent bond
with the Ser residue in the active site of the proteinase. The process is associated with
considerable conformational changes of the reacting molecules, which slows down the
rate of deacylation and results in the formation of stable inhibitor–enzyme complexes

3. Phytocystatins: Inhibitors of cysteine proteinases, acting on enzymes of the papain

family, are the best studied among plant proteinase inhibitors of other families. The
majority of proteins of this family are represented by members of the cystatin
superfamily (within which they constitute a specific family of phytocystatins). The
molecule of each phytocystatin contains all three structural elements involved in the
formation of complexes with enzymes in animal cystatins. These elements include the
residue Gly, which is located in the N-terminal portion of the molecule, the conserved
amino acid sequence Gln–X–Val–X–Gly, and the residue Trp in the C-terminal portion
of the molecule. The conserved amino acid sequence Gln–X–Val–X–Gly seems to make
the major contribution to the formation of the complex with a susceptible proteinase. This
conclusion is supported by the observation that phytocystatins devoid of Gly and Trp
residues in appropriate positions may retain relatively high inhibitory activities. NMR
studies of the spatial structure of rice cystatin (oryzacystatin I) demonstrated that all three
elements involved in the formation of the complex with the enzyme take the same
positions as in egg white cystatin. In addition, phytocystatins contain another conserved
sequence, Leu–Ala–Arg–Phe–Ala–Val–X–Glu–His–Asn, in the vicinity of the N-
terminal α-helix, which is absent from cystatins of animal origin. The role of this
sequence has not been clarified thus far. Its presence may reflect ancillary functions
played by cystatins in plants, which may have no immediate relation to effects on
proteinases.

4. Mustard trypsin inhibitor family: The seeds of cruciferous plants (Cruciferae) contain
thermostable low molecular weight trypsin inhibitors. In the seed of rape (Brassica napus
L.), these inhibitors account for 60–85% of total trypsin-inhibiting activity. The first
inhibitor to be obtained with a high degree of purity (MTI-2) was isolated from the seed
of white mustard (Sinapis alba L.). MTI-2 has a molecular weight of 7 kDa; its molecule
contains 63 amino acids, including eight Cys residues forming four disulfide bonds. An
inhibitor with closely similar properties (RTI) was isolated from the seed of oil rape
(Brassica napus (L.) var. oleifera). MTI-2 and RTI were shown to have homologous
primary structures (70% amino acid residues are shared), which differed from any other
structures of plant inhibitors of serine proteinases known at that time. Subsequent studies
demonstrated the presence of several forms of the inhibitors in rape and mustard seeds

5. Family of trypsin and α-amylase inhibitors from cereal grains: Trypsin inhibitors,
which account for 5−10% of total water-soluble protein in barley, wheat, and rye seeds,
are classified with two different families. The inhibitors of the germ portion of caryopsis
belong to the Baumann–Birk family (described above), whereas the inhibitors contained
in the endosperm constitute a separate family, which embraces the inhibitors of trypsin
and heterologous α-amylases. Trypsin inhibitors of these two types differ in both the
location within the caryopsis and the behavior throughout seed development and
germination

6. Potato inhibitor I family: Chymotrypsin inhibitor I was isolated from potato tubers and
crystallized in 1963. In addition to chymotrypsin, this protein acts on microbial serine
proteinases and inhibits trypsin (even though weakly). The inhibitor has a molecular
weight of 41 kDa; it is an oligomer composed of five structurally similar protomers.

7. Potato inhibitor II family: The first representatives of this family were isolated from
potato tubers and tomato leaves. These proteins act as potent inhibitors of chymotrypsin
and subtilisin, their effects on trypsin being less pronounced. The molecular weight of
each inhibitor approximates 21 kDa; the molecules are dimers of similar but not
completely identical subunits.