Structure and Function of Proteases

Cysteine proteases

In these enzymes, a cysteine residue, activated by a histidine residue, plays the role of the

nucleophile that attacks the peptide bond

Cysteine proteases are commonly encountered in fruits including the papaya, pineapple, fig

and kiwifruit. The proportion of protease tends to be higher when the fruit is unripe.

Cysteine proteases are used as an ingredient in meat tenderizers.

Papain in complex with its

covalent inhibitor E-64 (PDB id:

1PE6)

Examples of cysteine proteases: Papain, Cathepsins, Caspases, Actinidain, Bromelain,

Calpains, TEV protease

Cleavage site: [AVLIFWY]-[KR]-|-{V}

Papain also cleave the Fc portion of

immunoglobulins from the Fab portion.

Optimal temp: 60-70 C.

Mammalian proteases cathepsins have a role in the immune and other systems.

Activated at the low pH found in

lysosomes.

Cathepsins have a vital role in

mammalian cellular turnover, e.g.

bone resorption.

Caspases: play a major role in apoptosis. Active site is similar to that of papain, but their

overall structures are unrelated.

Substrate binding requires breaking a salt

bridge

Catalytic mechanism of cysteine proteases

Biological importance of cysteine proteases

In plants, cysteine proteases play multi-faceted roles such as physiology and development,

signalling pathways, senescence, apoptosis, storage of proteins in seeds, biotic and abiotic

stresses.

In humans, they are responsible for apoptosis, MHC class II immune responses, prohormone

processing, extracellular matrix remodeling, inflammation.

In traditional medicines, these are used to treat intestinal worm infections.

Aspartyl proteases

One aspartic acid residue (deprotonated) activates the attacking water molecule by poising it

for deprotonation, whereas the other aspartic acid residue (protonated) polarizes the peptide

carbonyl, increasing its susceptibility to attack.

Aspartyl proteases cleave preferably dipeptide bonds that have hydrophobic residues as well

as a β-methylene group.

Members of this class include renin, pepsin and HIV protease.

Why do two aspartic acids show different protonation states?

HIV protease: an unfused dimeric aspartyl protease that is similar to the fused protein.

Proposed catalytic mechanism for aspartic proteases

Unlike the closely related serine proteases these proteases do not form a covalent intermediate

during cleavage.

Metalloproteases

The active site of such a protein contains a bound metal ion, almost always zinc, that activates

a water molecule to act as a nucleophile to attack the peptide carbonyl group.

The ligands co-ordinating the metal ion can vary with histidine, glutamate, aspartate, lysine,

and arginine. The fourth coordination position is taken up by a labile water molecule.

Examples: thermolysin and meltrin. Thermolysin specifically catalyzes the hydrolysis of

peptide bonds containing hydrophobic amino acids.

Thermolysin is also widely used for peptide bond formation through the reverse reaction of

hydrolysis.

The digestive enzyme carboxypeptidase A is another classic examples of the zinc proteases.

Hydrolyzes carboxyl terminal peptide bond (Prefer bulky and aliphatic residues)

Structure and active site of carboxypeptidase A

Catalytic mechanism of carboxypeptidase A

Matrix metalloproteinases catalyze the reactions in tissue remodeling and degradation.

sp: signal sequence; pro: pro-domain; cat: catalytic domain; FNII: fibronectin type II motif; L1&L2:

linkers; Hpx: hemopexin domain; Mb: plasma membrane; TM: transmembrane domain; Cy: cytoplasmic

tail; CysR: cysteine rich; Ig: immunoglobulin domain; GPI: glycosylphosphatidilyinositol anchor.

Reaction pathway investigated for the peptide bond cleavage by MMPs

An isopeptide bond is an amide bond which forms between the carboxyl terminus of one

protein and the amino group of a lysine residue on another (target) protein.

Isopeptide bonds can occur between the side

chain amine of lysine and the side chain

carboxyl groups of either glutamate or

aspartate.

Bond formation can be (1) enzyme catalyzed (transglutaminases), (2) Spontaneous (HK97

bacteriophage capsid formation and Gram-positive bacterial pili).

Examples of isopeptide bond: glutathione, ubiquitin.

Deubiquitinating enzyme

Biological Roles of Isopeptide Bonds:

Signalling: influencing protein function, chromatin condensation, or protein half-life).

Structural: blood clotting, ECM modeling, apoptosis pathway, formation of pathogenic pilin,

actin skeleton remodeling.

Applications of spontaneous isopeptide bond formation

Peptide tag called SpyTag can spontaneously and irreversibly react with its binding partner

(SpyCatcher) through a covalent isopeptide bond.

The molecular tool can be used for in vivo protein targeting, fluorescent microscopy and

irreversible attachment for a protein microarray.

Deubiquitinating enzymes (DUBs) are a large group of proteases that cleave ubiquitin from

proteins and other molecules.

In humans there are nearly 100 DUB genes, which can be classified into two main classes:

cysteine proteases and metalloproteases.

These ubiquitin modifications are added to proteins by the ubiquitination machinary;

ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s) and ubiquitin

ligases (E3s).

Mechanisms of protease regulation

Protease

regulation

Gene expression,

activation of

zymogens

Targeting to

lysosomes

Regulation by post-

translational

modification

Endogenous inhibitors, Self-

cleavage

A total of 183 genes encoding protease inhibitors have been annotated in the rat genome,

which markedly contrasts with the more than 600 protease genes present in this species.

Protease inhibitors are important drugs

Protease inhibitors classification according to their mechanism of inhibition

1. Canonical inhibitors: In a virtually substrate-like manner (e.g. serpins).

2. Exosite-binding inhibitors: Binding to the adjacent region of the active site (e.g.

cystatins) .

3. TIMPs (tissue inhibitors of metalloproteinases): combination of the canonical and exosite

binding mechanisms.

4. Allosteric inhibitors: bind a region that is distantly located from the active site (e.g. X-

linked inhibitor of apoptosis protein, a caspase inhibitor).

Serpin superfamily: α1-antitrypsin, C1-inhibitor, antithrombin, α1-antichymotrypsin,

plasminogen activator inhibitor-1 and neuroserpin.

Most natural protease inhibitors are similar in structure to the peptide substrates of the enzyme

that each inhibits.

Renin-angiotensin-aldosterone system

Angiotensinogen

Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile-...

Angiotensin I

Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu | Val-Ile-...

Angiotensin II

Asp-Arg-Val-Tyr-Ile-His-Pro-Phe | His-Leu

Angiotensin III

Asp | Arg-Val-Tyr-Ile-His-Pro-Phe

Angiotensin IV

Arg | Val-Tyr-Ile-His-Pro-Phe

Captopril, an inhibitor of the metalloprotease angiotensin-converting enzyme (ACE)

Crixivan, an inhibitor of the HIV protease, is used in the treatment of AIDS

Structure of pepstatin in the binding pocket of pepsin

Pepstatin: aspartyl proteases inhibitor

Isovaleryl-Val-Val-Sta-Ala-Sta

Exercise:

List out the names of protease inhibitors and their target proteins routinely used in the

laboratory.