Structure and Function of Proteases
Protein Scissors
Proteins are tough, so we use an arsenal of enzymes to digest them into their component
amino acids.
Digestion of proteins begins in the stomach where hydrochloric acid unfolds proteins and the
enzyme pepsin begins a rough disassembly.
The real work then starts in the intestines. Enzymes on the surfaces of intestinal cells and
inside the cells chop them into amino acids ready for use throughout the body.
Proteases
action
Regulation and
localization of proteins
Activity of proteins, processing
of cellular information
Modulate protein
protein interactions
Create bioactive
molecules and
transduce signals
A protease is an enzyme catalyzes the hydrolysis of the peptide bonds in the polypeptide
chain of proteins.
Protease
influence
Heat shock and
unfolded protein
responses
Angiogenesis and
neurogenesis
Ovulation
fertilization, stem
cell mobilization
Immunity, necrosis, apoptosis,
tissue morphogenesis
Protease Function
Likewise, many infectious microorganisms require proteases for replication or use proteases
as virulence factors.
In plants, proteases contribute to the processing, maturation, destruction of specific sets of
proteins in response to developmental cues or to variations in environmental conditions.
Finally, proteases are also important tools of the biotechnological industry because of their
usefulness as biochemical reagents or in the manufacture of numerous products.
Proteases ranges from small enzymes (20 kDa) to sophisticated protein-processing and
degradation machines (700 to 6000 kDa).
Proteases can either break specific peptide bonds (limited proteolysis e.g. angiotensin-
converting enzyme), or break down a complete peptide to amino acids (unlimited proteolysis
e.g. proteinase K).
The vast proteolytic landscape
A large group of enzymes known as DUBs (deubiquitylating enzymes) can hydrolyze
isopeptide bonds in ubiquitin and ubiquitin-like protein conjugates.
Intramolecular autoproteases (such as nucleoporin and polycystin-1) hydrolyze only a single
bond on their own polypeptide chain but then lose their proteolytic activity.
Can proteases synthesize peptide bonds.
Exoproteases
Some of the proteases detach the terminal amino acids from the protein chain (exopeptidases
e.g. aminopeptidases, carboxypeptidase A).
Classification of proteases
1. Based on the peptide bond cleavage site
Endoproteases
Proteases cleaving the peptide bonds in the middle of a protein chain (endopeptidases e.g.
trypsin, chymotrypsin, pepsin, papain, elastase).
What is the difference between proteases and proteinases?
Serine proteases
These enzymes cleave the peptide bonds in proteins in which
serine serves as the nucleophilic amino acid at the active site.
2. Based on the character of catalytic active site
Cysteine (thiol) proteases
These enzymes degrade proteins in which cysteine is
involved in a nucleophilic attack.
Threonine proteases
These proteases are a family of proteolytic enzymes
harbouring a threonine (Thr) residue within the active site.
Glutamic acid proteases
These type protease have glutamic acid and glutamine
at their active site.
Metalloproteases
A protease which require a metal ion for its catalytic action.
Aspartic proteases
These proteases use an aspartate residue for catalysis of
their peptide substrates.
3. By optimal pH
Acid proteases
e.g. pepsin (aspartate proteases)
Basic proteases (or alkaline proteases)
e.g. subtilisin (serine protease)
Neutral proteases
e.g. calpains (Ca2+-dependent cysteine protease)
Catalytic class
Aspartic Cysteine Metallo Serine Threonine Total
Human 21 161 191 178 27 578
Mouse 27 178 206 227 26 664
Rat 24 173 200 226 29 652
According to Degradome database
According to MEROPS database
Catalytic class
Aspartic Cysteine Metallo Serine Threonine Glutamic Total
18576 90153 141384 136164 14958 383 401618
Distribution of proteases
Global view of the proteolytic landscape in representative eukaryotic genomes
Activation of proteases
Proteases are synthesized in the pancreas by protein biosynthesis as a precursor called
zymogen that is enzymatically inactive.
When it enters the intestine, the enzyme
enteropeptidase makes one cut in the trypsin chain,
clipping off the little tail.
Out body secretes 20-30 grams of digestive proteins, which are themselves digested when
they finish their duties.
Whether a protease can digest another protease molecule of its own type?
Dead intestinal cells and proteins leaking out of blood vessels are also digested and
reabsorbed as amino acids showing that our bodies are experts at recycling.
How many times a protease molecule can be used to digest a substrate protein molecule?
Serine proteases
Enzyme Source Function
Trypsin Pancreas Digestion of proteins
Chymotrypsin Pancreas Digestion of proteins
Subtilisin Bacillus subtilis Possibly digestion
Elastase Pancreas Digestion of proteins
Thrombin Vertebrate serum Blood clotting
Plasmin Vertebrate serum Dissolution of blood clots
Kallikrein Blood and tissues Control of blood flow
Complement C1 Serum Cell lysis in the immune response
Acrosomal protease Sperm acrosome Penetration of ovum
Lysosomal protease Animal cells Cell protein turnover
Cocoonase Moth larvae Dissolution of cocoon after metamorphosis
a-Lytic protease Bacillus sorangium Possibly digestion
Protease A and B Streptomyces griseus Possibly digestion
Trypsin Chymotrypsin Subtilisin
Broadly serine proteases can be classified based on their structures as: trypsin-like
(chymotrypsin-like) and subtilisin-like.
Convergent evolution of protease active sites
How does this arrangement of residues lead to the high reactivity of serine 195?
The histidine residue serves to position the serine side chain and to polarize its hydroxyl group
and acts as a general base catalyst (a hydrogen ion acceptor).
The aspartate residue helps orient the histidine residue and make it a better proton acceptor
through electrostatic effects.
Active site structure of a serine protease
Catalytic triads and hydrolytic enzymes
Catalytic triad was discovered in chymotrypsin. Based on these, other homologs such as
trypsin and elastase were found. The sequence identity among these enzymes are only about
40% with that of chymotrypsin, however, their overall structures are nearly the same.
Though these proteins operate by mechanisms identical
with that of chymotrypsin.
However, they have very different substrate specificities.
Trypsin cleaves at the peptide bond after residues with long,
positively charged side chains namely arginine and lysine.
Elastase cleaves at the peptide bond after amino acids with
small side chains such as alanine and serine.
Specificity of binding
Structure of chymotrypsin
Specificity: Peptide bond on carboxyl side of aromatic side chains (Y, W, F) & Large
hydrophobic residues (Met,…).
A clue came from the fact that chymotrypsin contains an extraordinarily reactive serine
residue. Treatment with organofluorophosphates such as diisopropylphosphofluoridate (DIPF)
was found to inactivate the enzyme irreversibly.
What is the nucleophile that chymotrypsin employs to attack the substrate carbonyl
group?
But the enzyme contains 28 serine residues.
The active site residues of chymotrypsin
Transition state stabilization: The Michaelis
complex
Transition state stabilization: The
tetrahedral intermediate
The structure stabilizes the tetrahedral intermediate of the chymotrypsin reaction.
The catalytic triad and oxyanion hole of chymotrypsin and subtilisin
Catalytic Mechanism of Chymotrypsin
Specificity nomenclature for protease-substrate interactions
The specificity of chymotrypsin depends amino acid on the amino-terminal side of the peptide
bond to be cleaved.
Residues on the amino-terminal side of the scissile bond: P1, P2, P3 and so forth.
Residues on the carboxyl side of the scissile bond: P1’ , P2’ , P3’ and so forth.
The corresponding sites on the enzyme are referred to as: S1, S2 or S1’ , S2’ and so forth.
Exercise:
List out all the names and classes of proteases which you use in the laboratory regularly.
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