3. Peptide bond is stable under
physiological conditions.
Half life = 100 years ,despite
thermodynamic instability
Hydrolysed under hash conditions
In acid : (HCL , 6M, 110C ,24-72 h )
Or base :( KOH ,4.2M ,100C ,24 h )
Scissile
bond
3
5. A protease performs Proteolysis
complete degradation of proteins to free amino acids.
not only in the digestive tract, but also in every cell in lysosomes, in
cytoplasm and other parts of the cell
is a ubiquitous mechanism the cell employs to regulate the function
and fate of proteins.
requires series of enzymes with different specificity.
5
6. Exopeptidases and Endopeptidases
(position of the cleavage site within the substrate molecule)
Exopeptidases
catalyzes the removal of
an amino acid at the end
of the polypeptide chain
releases a single amino
acid or dipeptide from
the peptide chain
Endopeptidases
catalyze the breaking of a
peptide bond within the
poplypeptide chain.
cannot break down peptides
into monomers.
6
8. Exopeptidases
1) aminopeptidases – cleave off a single amino acid from amino
terminus
2) dipeptidyldipeptidases – cleave off dipeptide from amino terminus
3) carboxypeptidases – cleave off a single amino acid from carboxy
terminus
4) dipeptidases – hydrolyse dipeptides in two single amino acids
8
9. Endopeptidases
Trypsin - cuts after Arg or Lys, unless followed by Pro.
Chymotrypsin - cuts after Phe, Trp, or Tyr, unless followed by
Pro.
Elastase - cuts after Ala, Gly, Ser, or Val, unless followed by
Pro.
Thermolysin - cuts before Ile, Met, Phe, Trp, Tyr, or Val,
unless preceded by Pro. Sometimes cuts after Ala, Asp, His or
Thr. Heat stable.
Pepsin - cuts before Leu, Phe, Trp or Tyr, unless preceded by
Pro. Also others,
Endopeptidase V8 cuts after Glu. (glutamate)
9
10. Classes of proteases
Class Nucleophile used Example
Serine Serine alcohol Trypsin ,thrombine,
elastase
Threonine Threonine secondary
alcohol
proteasome
cysteine Cysteine thiol Papaine, cathepsines
aspartyl Aspartate carboxyllic
acid
Pepsin , AIDS virus
protease
Glutamic Not found in
mammals
-
metalloproteases Zinc Carboxypeptidases,
botulinum and
tetanus toxins
10
12. Functions
Mediate processes that are frequently irreversible:
maturation of cytokines and prohormones.
breakdown of intracellular proteins.
regulate the fate, localization, and activity of many proteins.
modulate protein-protein interactions, create new bioactive molecules,
contribute to the processing of cellular information
remodeling, heat shock and unfolded protein responses,
12
16. specificity
most proteases are relatively nonspecific for substrates.
(subtilisin ).
some target multiple substrates in an indiscriminate manner
(e.g. proteinase K).
some highly specific and only cleave substrates with a certain
sequence. Blood clotting (such as thrombin) and viral polyprotein
processing (such as TEV protease).
16
17. Protease assay
Substrate :
casein
•Folin & Ciocalteus Phenol, or Folin’s reagent: reacts with free tyrosine
to produce a blue colored chromophore.
•Quantified and measured using a spec-660nm
• more tyrosine released from casein, more chromophores are
generated and the stronger the activity of the protease.
•Draw standard curve .
17
A protease (also termed 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 the polypeptide chain forming the protein.
Proteases can be found in animals, plants, bacteria, archea and viruses.
Proteolysis
complete degradation of proteins to free amino acids
requires series of enzymes with different specificity
not only in the digestive tract, but also in every cell in lysosomes, in cytoplasm and other parts of the cell
Aminopeptidase
Alanine
Arginyl
Aspartyl
Cystinyl
Leucyl
Glutamyl
Methionyl
3.4.13Dipeptidase
1
2
3
3.4.14Dipeptidyl peptidase
Cathepsin C
Dipeptidyl peptidase-4
Tripeptidyl peptidase
Tripeptidyl peptidase I
Tripeptidyl peptidase II
3.4.15Angiotensin-converting enzyme
3.4.16Serine type carboxypeptidases: Cathepsin A
DD-transpeptidase
3.4.17Metallocarboxypeptidases: Carboxypeptidase
Glutamate II
Other/ungroupedMetalloexopeptidase
Catalysis is achieved by one of two mechanisms:
Aspartic, glutamic and metallo proteases activate a water molecule which performs a nucleophilic attack on the peptide bond to hydrolyse it.
Serine, threonine and cysteine proteases use a nucleophilic residue in a (usually in a catalytic triad). That residue performs a nucleophilc attack to covalently link the protease to the substrate protein, releasing the first half of the product. This covalent acyl-enzyme intermediate is then hydrolysed by activated water to complete catalysis by releasing the second half of the product and regenerating the free enzyme
Thus, proteases regulate the fate, localization, and activity of many proteins, modulate protein-protein interactions, create new bioactive molecules, contribute to the processing of cellular information, and generate, transduce, and amplify molecular signals. As a direct result of these multiple actions, proteases influence DNA replication and transcription, cell proliferation and differentiation, tissue morphogenesis and remodeling, heat shock and unfolded protein responses, angiogenesis, neurogenesis, ovulation, fertilization, wound repair, stem cell mobilization, hemostasis, blood coagulation, inflammation, immunity, autophagy, senescence, necrosis, and apoptosis.
some proteases exhibit an exquisite specificity toward a unique peptide bond of a single protein (e.g. angiotensin-converting enzyme);
most proteases are relatively nonspecific for substrates.
some are overtly promiscuous and target multiple substrates in an indiscriminate manner (e.g. proteinase K).
Proteases also follow different strategies to establish their appropriate location in the cellular geography and, in most cases, operate in the context of complex networks comprising distinct proteases, substrates, cofactors, inhibitors, adaptors, receptors, and binding proteins, which provide an additional level of interest but also complexity to the study of proteolytic enzymes.
Black microplates are pre-coated with protease-specific antibody. The sample is added allowing both pro- and active forms of the enzyme to bind the immobilized antibody. Unbound proteases are removed following wash steps.
A protease-specific peptide substrate (green & purple) is added. The substrate features a fluorophore (green) and quencher molecule (purple) on opposite sides of the prospective cleavage site.
Active enzyme cleaves the peptide substrate between the fluorophore and the quencher molecule, increasing the distance between them. Energy from the fluorophore is now available as fluorometric signal since the quencher is no longer close enough to absorb it. The resulting signal is directly proportional to the amount of active protease bound in the initial step.