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.