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Proteolytic Enzyme: Basic information

and cleavage rules
Dr. Aditya Arya
Date of publication: 10 June 2016

Proteolytic enzymes are commonly used to determine the sequence of small protein sequences. Most
of the proteolytic enzymes are capable of cleaving the protein at specific positions. Cleaving a protein
with specific enzyme and then analysing the fragments may help in the determination of the order of
amino acids by simple logical reasoning. Past years trends of CSIR-NET exam and other nationwide
competitive exams have shown frequent usage of these rule in questions. The question might appear
very difficult and cannot be solved if we do not know the cleavage rules. In this quick note we will
understand how to solve such questions and also a detailed view cleavage rules. This note has been
prepared by content provided by global experts in the field of proteins (European Molecular Biology
Laboratory and Swiss Bioinformatics Institute).

INTRODUCTION
A protease (also called a peptidase or proteinase) is any enzyme that performs proteolysis, that is, begins
protein catabolism by hydrolysis of the peptide bonds that link amino acids together in a polypeptide
chain. Proteases have evolved multiple times, and different classes of protease can perform the same
reaction by completely different catalytic mechanisms. Proteases can be found in animals, plants,
bacteria, archaea and viruses.
In plants, their genome encodes for hundreds of proteases, largely of unknown function. Those with
known function are largely involved in developmental regulation. Plant proteases also play a role in
regulation of photosynthesis
In animals proteases are used throughout an organism for various metabolic processes. Acid proteases
secreted into the stomach (such as pepsin) and serine proteases present in duodenum (trypsin and
chymotrypsin) enable us to digest the protein in food. Proteases present in blood serum (thrombin,
plasmin, Hageman factor, etc.) play important role in blood-clotting, as well as lysis of the clots, and the
correct action of the immune system. Other proteases are present in leukocytes (elastase, cathepsin G)
and play several different roles in metabolic control. Some snake venoms are also proteases, such as pit
viper haemotoxin and interfere with the victim's blood clotting cascade.
Bacteria secrete proteases to hydrolyze (digest) the peptide bonds in proteins and therefore break the
proteins down into their constituent monomers (amino acids). Bacterial and fungal proteases are
particularly important to the global carbon and nitrogen cycles in the recycling of proteins, and such
activity tends to be regulated by nutritional signals in these organisms.

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Some viruses express their entire genome as one massive polyprotein and use a protease to cleave this
into functional units (e.g. polio, norovirus, and TEV proteases). These proteases (e.g. TEV protease) have
high specificity and only cleave very restricted set of substrate sequences.
Recent trend of competitive exam had shown recurrent questions from this topic, including many questions on
determine the sequence of peptide or name and classes of proteases. This note will enable us to solve such questions.

CLASSIFICATION OF PROTEASES
Proteases were first grouped into 84 families according to their evolutionary relationship in 1993, and
classified under four catalytic types: serine, cysteine, aspartic, and metallo-proteases. The threonine and
glutamic-acid proteases were not described until 1995 and 2004 respectively. The mechanism used to
cleave a peptide bond involves making an amino acid residue that has the cysteine and threonine
(proteases) or a water molecule (aspartic acid, metallo- and glutamic acid proteases) nucleophilic so that
it can attack the peptide carboxyl group. One way to make a nucleophile is by a catalytic triad, where a
histidine residue is used to activate serine, cysteine, or threonine as a nucleophile. This is not an
evolutionary grouping, however, as the nucleophile types have evolved convergently in different
superfamilies, and some superfamilies show divergent evolution to multiple different nucleophiles.
Proteases can be classified into seven broad groups (Kohei Oda, 2012)

Serine proteases - using a serine alcohol

Examples: Trypsin, Chymotrypsin, Thrombin, Elastase, Subtilisin
Cysteine proteases - using a cysteine thiol
Examples: Papain, Caspase1, TEV protease, Calpain, hepatitis virus protease
Threonine proteases - using a threonine secondary alcohol
Examples: ornithine acetyltransferase, Archean protosome
Aspartic proteases - using an aspartate carboxylic acid
Examples: Pepsin, Cathepsin, Renin, HIV protease, Presenillin, bacterial type 4 prepilin
Glutamic proteases - using a glutamate carboxylic acid
Examples: Eqolisin (from : Scytalidium lignicola), aspergilloglutamic peptidase
Metalloproteases - using a metal, usually zinc and rarely cobalt as cofactor
Examples: Exopeptidases: carboxypeptidases , metalloexopeptidases,
Endopeptidases: ADAM proteins and matrix metalloproteinases.
Asparagine peptide lyases - using an asparagine to perform an elimination reaction (not
requiring water)
Examples: Tsh protein precursor of Escherichia coli

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CLEAVAGE BY PROTEASES AND PROBLEM SOLVING

Although there are hundreds of proteases and their selectivity towards the cleavage of a peptide bond
varies. In the next section we will see in detail the cleavage rules of most of the proteolytic enzyme but
before that let have a glimpse of most commonly used enzymes and their cleavage rules.

Sr. No.

Name of Enzyme

Cleavage Rule

ENDOPEPTIDASE (cleave internal peptide linkages)

1.
Trypsin
Cleaves after (at the C terminal of ) Arg or Lys (Basic AA)
2.
Chymotrypsin
Cleaves after Phe,Trp, Tyr (aromatic AA)
3.
Pepsin
Cleaves after Phe or Leu
4.
Elastase
Cleaves after Gly, Ala, Val, Ser
5.
Endoproteinase Lys-C
Cleaves after Lys
6.
Endopeptidase V8 or Glu-C
Cleaves after Glu
7.
Thermolysin
Cleaves before (at N ter) Ile, Met, Phe, Trp, Tyr or Val
EXOPEPTIDASE (cleave external /terminal peptide linkages)
8.
Carbonxypeptidase A
Removes C-terminal AA (NOT Arg or Lys) A for Aromatic
9.
Carbonxypeptidase B
Removes C-terminal AA (ONLY Arg or Lys) B for basic AA
*No cleavage if any target residue is followed by Proline

Apart from the proteolytic enzymes some chemicals are also specific in their cleavage, which also prove
helpful in determing the order of amino acids in a given peptide. Following are such non-enzyme reagents
which show selective cleavage

Sr. No.
1.
2.
3.
4.
5

Name of Enzyme
Cyanogen Bromide (CNBr)
Formic Acid
N-Bromosuccinimide (NBS)
N-Bromosuccinimide (NCS)
BNPS Skatole

Cleavage Rule
Cleaves after Met
Cleaves after Asp
Cleaves after Trp
Cleaves after Trp
Cleaves after Tyr

While solving questions on cleavage rules we must keep in mind that:

Conventionally a protein sequence is written from N terminal to C terminal hence, a

proteolytic enzyme causing cleavage of the protein may be represented by N- terminal side of
a residue or C-terminal side of a residue.
A cleavage rule may fail if there are some residues which hinder the recognition of cleavage
site (which are described as exceptions such as presence of proline before recognition
residue).
Usually peptides are linear, but in case of circular peptides the number of fragments formed
will be one lesser than those formed in linear peptide by same number of cleavage

The detailed description of Cleavage rules of proteases is given below:

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DETAILED CLEAVAGE RULES:

The following enzymes potentially cleave when the respective compositions of the cleavage sites are found. However, there also are
some exceptions. Before reading the following table carefully understand the P1P4 , P1 P4 notations from the above figure.
Enzyme name
Arg-C proteinase
Asp-N endopeptidase
BNPS-Skatole
Caspase 1
Caspase 2
Caspase 3
Caspase 4
Caspase 5
Caspase 6
Caspase 7
Caspase 8
Caspase 9
Caspase 10
Chymotrypsin-high specificity (Cterm to [FYW], not before P)
Chymotrypsin-low specificity (Cterm to [FYWML], not before P)

P4
F, W, Y, or L
D
D
L
L or W
V
D
I or L
L
I
-

P3
V
M
E
E
E
E
E
E
E
-

P2
H, A or T
A
Q
V
H
H or I
V
T
H
A
-

P1
R
W
D
D
D
D
D
D
D
D
D
D
F or Y
W
F,L or Y
W

P1'
D
not P, E, D, Q, K or R
not P, E, D, Q, K or R
not P, E, D, Q, K or R
not P, E, D, Q, K or R
not P, E, D, Q, K or R
not P, E, D, Q, K or R
not P, E, D, Q, K or R
not P
not M or P
not P
not M or P

P2'
-

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Clostripain (Clostridiopeptidase B)
CNBr
Enterokinase
Factor Xa
Formic acid
Glutamyl endopeptidase
GranzymeB
Hydroxylamine
Iodosobenzoic acid
LysC
Neutrophil elastase
NTCB (2-nitro-5-thiocyanobenzoic
acid)
Pepsin (pH1.3)
Pepsin (pH>2)
Proline-endopeptidase
Proteinase K
Staphylococcal peptidase I
Thermolysin
Thrombin

Trypsin (please note

the exceptions!)
Enzyme name

D or E
A,F,G,I,L,T,V or M
I
-

D or E
D or E
E
-

D or E
G
P
-

M
H
R
M
K
R
D
E
D
N
W
K
A or V
-

not P or Y
not D,M,P or W
G
C

A,F,G,I,L,T,V or M

not H,K, or R
not H,K, or R
not H,K or R
not H,K or R
A,F,G,I,L,T,V,W
or A
P3

not P
not P
not P
not P
H,K or R
not E
G
P

not R
F or L
not R
F,L,W or Y
P
A,E,F,I,L,T,V,W or Y
E
not D or E
R
R

F or L
F,L,W or Y
not P
A,F,I,L,M or V
G
not D or E

not P
not P
not P
not P
not DE

W
M
P2

K or R
K
R
P1

not P
P
P
P1'

P2'

P4

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The exception rules:

The above cleavage rules do not apply, i.e. no cleavage occurs, with the following compositions of the
cleavage sites:
Enzyme name P4 P3 P2

Trypsin

P1 P1'

P2'

C or D K D

K H or Y -

R K

R H or R -

A first and obvious approach to obtain information concerning the cleavage specificity of a protease is the
characterization of the respective natural substrate. In a next step, standard polypeptides can be used for
digestion such as glucagon or insulin chains. However, an ideal polypeptide substrate should contain all
possible 400 combinations of dipeptide bonds, whereas in insulin and glucagon for example only small
fractions of these combinations can be found (not to mention the possibilities of tetrapeptides composition
when taking into account the sites P2 to P2'). Ideally, the cleavage data would be obtained by digesting all
available proteins in the databases, but this is beyond feasibility. A more systematic and complete approach
would be to test the proteases with substrates of low molecular weight such as di-, tri, tetrapeptides etc.
Unfortunately, the available data for most proteases is still very incomplete. Only for a few proteases
enough information has been accumulated that allow a statistical treatment (for details see Kiel, 1992)
resulting in a more complete and refined picture of cleavage specificity. In the following, specific cleavage
preferences of individual enzymes are reported. Only the accordingly derived rules are taken into
consideration by the program peptide Cutter. Thus, the user should be aware of the fact that results
obtained by experiments may differ from the predictions made by the Peptide Cutter program.

PREVIOUS QUESTIONS FROM CSIR-NET EXAM:

Q1. The Oligopeptide, F-A-R-P-M-T-S-R-P-G-R, is treated with trypsin, chymotrypsin and
carbonxypeptidase-B. apart from the original peptide, the number of fragments obtained will be: (CSIRNET June 2012]
1. 4
2. 3
3. 2
4. 0
Solution: Based on the above discussion, we may find here that trypsin cleaves a peptide after Arg or Lys,
here in this peptide, there is no lysine but three Arg, one of them is at terminal and other two are followed
by a proline, so considering the exception rule, there be no cleavage caused by trypsin. Chymotrypsin
cleaves the peptide at C terminal of F, W or Y, in this peptide there is only one F which is present at N
terminal so no cleavage by this enzyme as well. Lastly, carbonxypeptidase B cleaves after K or R at C
terminal, here in this sequence there is a terminal R that may be cleaved by this enzyme but it will not
create two fragments rather and amino acid and same fragment. So ideally 1 fragments will be correct,
which is not in the options. Hence the best possible answer is 2 fragments, option 3.

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Q2: In order to determine the primary structure of an octapeptide, amino acid composition was
determined by acid hydrolysis (A). The intact oligopeptide was treated with carboxypeptidase (B),
chymotrypsin (C), trypsin (D) and CNBr (E). The peptides were separated in each case and acid
hydrolysis was carried out for B.- E. Following results were obtained (the brackets represent
mixtures of amino acids in each fragment): [CSIR-NET JUNE 2013]
(A)
(2Ala, Arg, Lys, Met, Phe, 2Ser)
(B)
(Ala, Arg, Lys, Met, Phe, 2Ser) and Ala
(C)
(Ala, Arg, Phe, Ser), (Ala, Lys, Met, Ser)
(D)
(Ala, Arg) , (Lys, Phe, Ser) ,(Ala, Met, Ser)
(E)
(Ala, Arg, Lys, Met, Phe, Ser), (Ala, Ser)
Which one is the correct sequence of the oligopeptide?
1.
Arg Ala Ser Lys Met Phe Ser- Ala
2.
Arg Ala Ser Phe Lys Met - Ser Ala
3.
Ala Arg Ser Phe Lys Met Ser Ala
4.
Ala Arg Phe Ser Lys Met Ser Ala
Solution: Based on the above discussion, we learnt that peptide can be specifically cleaved by proteases
and then here, it was followed by hydrolysis causing separation of individual amino acids. This question
requires analytical reasoning. We may easily look at the sequence and try to rule out the possibilities
based on experimental observations.
In the first experiments results of hydrolysis merely suggest that there are two and 2 Ala but no clue is
obtained about their relative arrangement.
In Next experiment, carbonxypeptidase cleavage produced residues shown in option (B) where one Ala
was produced which indicates that Ala is present at C terminal, which is seen in all 4 options in the
question, so this does not solve the purpose.
In third experiment (C) cleavage by chymotrypsin will occur after aromatic amino acid, so in first set we
see only one aromatic amino acid (Phe). So option 1 and option 4 are ruled out.
Result shown in E and D may be compared to reveal one fact, As D represents cleavage by trypsin and E
represents cleavage by CNBr, cyanogen bromide cleaves only after methionine so Ala and Serine were
present after methionine (So rule out option 1) as this doesnt support the experiment results.
Cleavage by trypsin produced Ala, Arg which means the option 3 or 4 may be right, but option 4 is already
ruled out on the basis chymotrypsin cleavage.
Hence the option 3 is correct.

Finally we can tally the results of experiments given in the question by cross checking the answer and
cleavage sites.

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Q3: Consider the structure less oligopeptide, R G P S T-S -K M - P E - Y G - S T - D Q S N

- W H F R. The numbers of bonds that will be cleaved by trypsin and chymotrypsin treatments
separately, are: [CSIR- NET DEC 2013]
1. 1, 2
2. 2, 2
3. 2, 3
4. 1, 3
Solution: trypsin will cleave at C terminal of Lys (K) or Arg (R), provided they are not followed by proline,
highlighted positions in the sequence,:
R G P S T-S -K M - P E - Y G - S T - D Q S N - W H F R.
Chymotrypsin will cleave at C terminal of Phe (F), Trp (W) or Tyr (Y), provided they are not followed by
proline, highlighted positions in the sequence :
R G P S T - S - K M - P E - Y G - S T - D Q S N - W H F R.
Therefore the answer to this question is first - 1, 2 peptide bonds will be cleaved by trypsin and
chymotrypsin respectively.

Some more practice questions:

1. Amino acid analysis of an oligopeptide 7 residues long gave
Asp Leu Lys Met Phe Tyr
The following facts were observed:
a) Trypsin treatment had no apparent effect.
b) The PTH derivative released by Edman degradation was

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c) Brief chymotrypsin treatment yielded several peptides, including a dipeptide and a tetrapeptide. The
amino acid composition of the tetrapeptide was Leu, Lys, and Met.
d) Cyanogen bromide treatment yielded a dipeptide, a tetrapeptide, and free Lys.
What is the amino acid sequence of this peptide?
Q2. Amino acid analysis of a decapeptide revealed the presence of the following products:
Asp Asn Tyr Arg Met Cys Lys Ser Phe
The following facts were observed:
a) The c-terminal amino acid contained a sulfur atom.
b) Trypsin treatment yielded two tetrapeptides and a free Lys.
c) Clostripain treatment yielded a tetrapeptide and a hexapeptide. (Clostripain cleaves after arginine
residues only.)
d) Cyanogen bromide treatment yielded an octapeptide and a dipeptide of sequence NC
e) Chymotrypsin treatment yielded two tripeptides and a tetrapeptide. The N-terminal chymotryptic
peptide had a net charge of -1 at neutral pH and a net charge of -3 at pH 12.
f) One cycle of Edman degradation gave the PTH derivative:
What is the amino acid sequence of this peptide?
Q3. A hexapeptide contains the following amino acids:
Met Phe Lys Ser Trp Leu
Determine the amino acid sequence of the peptide using the following data:
a) One round of Edman degradation yielded the PTH derivative:
b) Treatment of the peptide with trypsin yielded a dipeptide with a +1 charge at physiological pH and a
neutral tetrapeptide.
c) Treatment of this peptide with cyanogens bromide yielded two tripeptides.
d) Treatment of this peptide with chymotrypsin yielded a free F and a pentapeptide.

Answers:
Q1. FDYMLMK

Q2. SDYRKKFMNC

Q3. SKMLWF

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CLINICAL APPLICATIONS OF PROTEASES.

Proteolytic enzymes are indicated in inflammatory conditions and to support the immune system.
Proteolytic enzymes (or proteases) refer to the various enzymes that digest (break down into smaller units)
protein. These enzymes include the pancreatic proteases chymotrypsin and trypsin, bromelain (pineapple
enzyme), papain (papaya enzyme), fungal proteases, and Serratia peptidase (the silk worm enzyme).
Preparations of proteolytic enzymes have been shown to be useful in the following situations:

Fig 1: Potential applications of proteases in industries (Image credit Li et al, FEBS letters, 2013 Volume 587 (8), 11551163)

A serine protease of human origin, activated protein C, was produced in recombinant form and marketed
as Drotrecogin alfa (also known as Xigris (TM)) and licensed for intensive-care treatment of severe sepsis. It
was voluntarily withdrawn by the manufacturer in 2011 after being shown to be ineffective.
Tissue plasminogen activator (TPA) is a serine protease occurring in animals including humans. Humanidentical TPA (produced industrially by genetically recombinant microorganisms) has an established
medical use in the treatment of ischemic stroke: by its proteolytic activity it enables the action of another
enzyme (plasmin), which breaks down the protein (fibrin) of blood clots.
Protease are one of the component (enzyme) in Sollpura (Liprotamase), an pancreatic enzyme replacement
therapy (PERT). It assists in the breakdown of proteins into amino acids and polypeptides.
Serratia E-15 protease (also known as serratiopeptidase or serrapeptidase, another protease that has been
proposed as an anti-inflammatory agent. It is also known at research level (not clinical lever yet) that Serratia
E-15 protease (also known as serratiopeptidase) was effective for eradicating infection caused by biofilmforming bacteria in an experimental animal model.

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Papain is a protease obtained from the latex of the fruit of the papaya tree. It has been used (without
regulation) for wound debridement for many years, but in the USA in 2008 it was brought under regulation
by the U.S. Food and Drug Administration and removed from sale for this purpose, following reports of
adverse effects. On the other hand, recent research has been exploring new ways of administering papain
for wound debridement.
Papain as well as other proteases, including bromelain, collagenase, trypsin and thermolysin, have also been
tried or used according to other reports on the use of proteases for debridement of wounds and burns
without damaging healthy tissue.
Maggot therapy (also known as maggot debridement therapy (MDT), larval therapy, larva therapy, larvae
therapy, biodebridement or biosurgery) is a type of biotherapy involving the introduction of live, disinfected
maggots (fly larvae) into the non-healing skin and soft tissue wound(s) of a human or animal for the purpose
of cleaning out the necrotic (dead) tissue within a wound (debridement) and disinfection. Maggots secrete
serine protease which helps in would debridement.
Nattokinase may be potentially useful as a clot-buster and blood thinner, and is considered by some in the
alternative medicine community as a possible adjunctive therapy in cardiovascular disease. Nattokinase is
an enzyme (EC 3.4.21.62) extracted and purified from a Japanese food called natt.
Some viruses express their entire genome as one massive polyprotein and use a protease to cleave this into
functional units (e.g. polio, norovirus, and TEV proteases). These proteases (e.g. TEV protease) have high
specificity and only cleave very restricted set of substrate sequences.
Digestive proteases are part of many laundry detergents and are also used extensively in the bread industry
in bread improver.
Protease inhibitors
The activity of proteases is inhibited by protease inhibitors.[14] One example of protease inhibitors is the
serpin superfamily, which includes alpha 1-antitrypsin, C1-inhibitor, antithrombin, alpha 1antichymotrypsin, plasminogen activator inhibitor-1, and neuroserpin.
Natural protease inhibitors include the family of lipocalin proteins, which play a role in cell regulation and
differentiation. Lipophilic ligands, attached to lipocalin proteins, have been found to possess tumor
protease inhibiting properties. The natural protease inhibitors are not to be confused with the protease
inhibitors used in antiretroviral therapy. Some viruses, with HIV/AIDS among them, depend on proteases
in their reproductive cycle. Thus, protease inhibitors are developed as antiviral means.

Reference, credits and Suggested readings:

1. http://web.expasy.org/peptide_cutter/peptidecutter_enzymes.html
2. https://www.ncbi.nlm.nih.gov/pubmed/22016395
3. https://en.wikipedia.org/wiki/Proteases_(medical_and_related_uses)
Readers are advised to refer two basic reviews for exploring commercial applications of proteases.
4. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4503466/pdf/nihms706169.pdf
5. http://www.sciencedirect.com/science/article/pii/S0014579313000082