Protease

Enzyme Nomenclature

• Add –ase to the name of the substrate

– Urease Phosphatase

• Class, subclass, subsubclass, serial no.

EC 3.4.21.5

    

The Six Classes of Enzymes

1. Oxidoreductases (dehydrogenases)

Catalyze oxidation-reduction reactions

2. Transferases

Catalyze group transfer reactions

3. Hydrolases

Catalyze hydrolysis reactions where water is the acceptor of the transferred group

4. Lyases

Catalyze lysis of a substrate, generating a double bond in a nonhydrolytic, nonoxidative elimination (Synthases catalyze the addition to a double bond, the reverse reaction of a lyase)

5. Isomerases

Catalyze isomerization reactions

6. Ligases (synthetases)- Catalyze ligation, or joining of two substrates- Require chemical energy (e.g. ATP)

Coenzyme

• Many enzymes require nonprotein components to carry out their catalytic function- cofactors

• Cofactors may be metal ions or organic molecules (coenzyme)

• Cofactor: metal ion + coenzyme

• Many coenzymes are vitamins or contain vitamins as part of their structure

• Tightly bound coenzymes are referred as prosthetic groups

Holoenzyme and Apoenzyme

• Holoenzyme– Complex of protein and prosthetic groups

– Catalytically active

• Apoenzyme– The enzyme without the prosthetic groups

– Catalytically inactive

Oxidation-reduction reactions

• Electrons are transferred between two species

• Oxidizing agent gains electrons (is reduced)

• Reducing agent donates electrons (is oxidized)

Acid-Base Catalysis

• Reaction acceleration is achieved by catalytic transfer of a proton

• A general base (B:) can act as a proton acceptor to remove protons from OH, NH, CH or other XH

General base catalysis reactions

• A general base (B:) can remove a proton from water and thereby generate the equivalent of OH- in neutral solution

• A general acid (BH+) can donate protons

Covalent Catalysis

• All or part of a substrate is bound covalently to the enzyme to form a reactive intermediate

• Group X can be transferred from A-X to B in two steps via the covalent ES complex X-E

A-X + E X-E + A

X-E + B B-X + E

Peptidase

Protease (Peptide hydrolase, EC 3.4.)

• Exopeptidase (EC 3.4.11-19)

– Act on N- or C-terminus of the peptide

• Endopeptidase (EC 3.4.21-24)

– Classification based on the catalytic residues in the active site

Endopeptidase

• Serine proteinases (EC 3.4.21)

• Cysteine proteinases (EC 3.4.22)

• Aspartic proteinases (EC 3.4.23)

• Metallo-proteinases (EC 3.4.24)

• EC 3.4.99– A new, temporary subclass– Unknown catalytic mechanism

Serine proteinases (EC 3.4.21)

• Contains serine at the active site

• Catalytic triad

• Covalent binding of substrate to Ser

Inhibitors of serine proteinases

• Inhibited by diisopropyl fluorophosphate (DIF, DIFP) and diisopropyl phosphofluoridate (DIPF)

• Most inhibited by phenylmethanesulfonyl fluoride (PMSF)

• Some inhibited by chloromethyl ketone– TLCK (N-p-tosyl-L-lysine chloromethyl ketone)– TPCK (L-1-tosylamido-2-phenylethyl chloromet

hyl ketone)

Inhibition of serine protease with DFP

• Diisopropyl fluorophosphate (DFP) is an organic phosphate that inactivates serine proteases

• DFP reacts with the active site serine (Ser-195) of chymotrypsin to form DFP-chymotrypsin

Organophosphorous inhibitors

• Such organophosphorous inhibitors are used as insecticides or for enzyme research

• These inhibitors are toxic because they inhibit acetylcholinesterase (a serine protease that hydrolyzes the neurotransmitter acetylcholine)

Serine proteinases (EC 3.4.21)

• Two superfamily– Chymotrypsin family

• Trypsin• Chymotrypsin• Elastase

• kallikrein and subtilism family• Subtilisin

Binding sites of chymotrypsin, trypsin, and elastase

• Substrate specificities are due to relatively small structural differences in active-site binding cavities

Serine Proteases

• -Chymotrypsin active site groups include:

Ser-195, His-57 , Asp-102

Catalytic triad of chymotrypsin

Cysteine proteinases

• Contains cysteine at the active site

• Covalent binding of substrate to Cys

• In cytosol or in lysosome

Inhibitors of cysteine proteinases

• Inhibited by low concentration of pHMB (p-hydroxymercuribenzoate), pCMB (the hydrolysis product of p-chloromercuribenzoate)

• Inhibited by alkylating agents– Iodoacetate– Iodoacetamide– N-ethylmaleimide

Inhibitors of cysteine proteinases

• Sulfhydryl reagents are not specific for Cys at the active site– Many proteinases contain –SH in their structure

s

• Active site-specific cysteine reagent– E-64 [L-trans-epoxysuccinyl-leucylamido (4-gu

anidino) butane]

Cysteine proteinases

• Papain- MW 23,000, a single polypeptide

• Lysosomal cysteine proteinase– Cathepsin B, H, L, S

• Calpain– A cytosolic enzyme

• Metal-dependent cysteine proteinase

Aspartic proteinases(Acid proteinases, EC 3.4.23)

• Contains Asp at active site

• General acid-base catalysis

• No formation of covalent enzyme-substrate complex

• Exists in secretory granules, membranes, endosomes, or lysosomes

• Present in eukaryotes, not in prokaryotes

Inhibitors of aspartic proteinases

• Inhibited by pepstatins

• Inhibited by diazoacetyl compounds– Diazoacetyl-L-phe-methyl ester

Aspartic proteinases

• Pepsin family – Digestive enzymes: pepsin and chymosin– Lysosomal cathepsins D– Processing enzymes: renin– Certain fungal proteases: penicillopepsin, rhizo

puspepsin, endothiapepsin• Viral proteinases

– Protease from the AIDS virus (HIV), also called retropepsin

Metallo-proteinases(EC 3.4.24)

• Contains metal ions at the active center

• The metal ions are an integral part of their structures, and enhance the nucleophilicity of H2O and polarize the peptide bond to be cleaved prior to nucleophiolic attack

Nucleophilic species are electron rich

Electrophilic species are electron poor

Many Enzymes Require Inorganic Cations

• Metal-activated enzymes

• Metalloenzymes

Metal-activated enzymes

• Have an absolute requirement or are stimulated by metal ions (examples: K+, Ca2+, Mg2+)

• Binds metals loosely

• Contains Cys or Ser at the active site

Metallo-enzymes

• Contain firmly bound metal ions at the enzyme active sites (examples: iron, zinc, copper, cobalt )

• Widely distrubuted in prokaryotes and eukaryotes

• Most of the exopeptidases are metallo-proteins

Metallo-proteinase

• Exists in ER, plasma membrane, mitochondria, cytosol

• Inhibited by chealting agents- EDTA, EGTA

• Metal chelator may inhibit metal-activated proteinases in addition to metallo-proteinases

Regulation of proteinases activity

• Compartmentation– Cathepsin in lysosome– Attachment of proteinases to membranes results in a los

s of freedom

• Synthesis and degradation• Inhibitors and activators• Regulation by metabolites

– Ca2+ , nucleotide• Covalent modification

Protease inhibitors

Leupeptin

• C20H38N6O4 (1/2 H2SO4(H2O)• Formula Weight: 493.62• A protease inhibitor, will strongly inhibit tr

ypsin, papain, plasmin, thrombokinase, kallikrein and cathepsin B.

• The half-maximal inhibitory concentration ranges from 0.5 to 75µg/ml, depending on the enzyme and the substrate.

• Leupeptin does not inhibit chymotrypsin, elastase, renin, or pepsin.

• Storage: -20°C

 Leupeptin

• Structure: Acetyl-leucyl-leucyl-arginal • Inhibition spectrum: inhibits serine (trypsin (Ki=

13 µM), plasmin, porcine kallikrein) and cysteine proteinases (papain, cathepsin B). Does not inhibit chymotrypsin and thrombin.

• Mechanism of action: Competitive and reversible inhibitor. Inhibition may be relieved by an excess of substrate.

• Properties: Soluble in water, ethanol, acetic acid and DMF (Stock solution: 10 mM)MW: leupeptin: 426.6; leupeptin hemisulphate monohydrate: 542.7

Suggested final concentration:

1-10 µM (0.5-1 µg/ml)

 Leupeptin

PMSF

• A widely used serine protease

• Half-life in water: 15-60 minutes (depending on your reference source).

• PMSF is not very soluble in water and should be kept at -20°C in dry methanol/propanol.

Proteases cocktail • PMSF

– Stock 100 mM (keep 4°C methanol),

– Use at 1 mM

– Inhibit serine proteases

• E64

– Stock 1 mM (keep -20°C, H20)

– Use at 10 mM

– Inhibit cysteine proteases (papain, calpain, cathepsin B, cathepsin L)

• EDTA

– Stock 500 mM (keep 4°C)

– Use at 5 mM

– Inhibit metallo-proteases

Leupeptin

• C20H38N6O4

• Acetyl-leucyl-leucyl-arginal

• Inhibitor of serine and cysteine proteases

http://www.bikaken.or.jp/compound/substfile/leupep.htm

Plenylmethylsulfonyl fluoride

• C7H7FO2S

• Irreversible inhibitor of serine proteases

http://chemdb2.niaid.nih.gov/struct_search/images/structures/043580.gif