PROTEASES

Done by : Apurva Fernandes

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CONTENTS
Peptide bonds
Proteolysis
Classes of proteases
Catalysis
Fucntions
Specificity
Assay
Refrences

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 Peptide bond is stable under
physiological conditions.
Half life = 100 years ,despite
Scissile
thermodynamic instability
bond

Hydrolysed under hash conditions

In acid : (HCL , 6M, 110C ,24-72 h )
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Or base :( KOH ,4.2M ,100C ,24 h )
Biological strategies: PROTEASES !!
also called Peptidases / proteinase

found in animals, plants, bacteria, archea and viruses

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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.

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Exopeptidases and Endopeptidases
(position of the cleavage site within the substrate molecule)

Exopeptidases Endopeptidases
catalyze the breaking of a
catalyzes the removal of
peptide bond within the
an amino acid at the end
poplypeptide chain.
of the polypeptide chain
cannot break down peptides
releases a single amino
into monomers.
acid or dipeptide from
the peptide chain

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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

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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)

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Classes of proteases
Class Nucleophile used Example

Serine Serine alcohol Trypsin ,thrombine,

elastase
Threonine Threonine secondary proteasome
alcohol

cysteine Cysteine thiol Papaine, cathepsines

aspartyl Aspartate carboxyllic Pepsin , AIDS virus

acid protease

Glutamic Not found in -

mammals

metalloproteases Zinc Carboxypeptidases,

botulinum
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and
tetanus toxins
Catalysis

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 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,

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 Functions..

Blood Coagulation .
Thrombin activates fibrinogen resulting in
fibrin , causing hemostatis.

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Functions ..

Digestion
Enzymes : Pepsin,
chymotrypsin ,
elastase .

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Functions..
Apoptosis
Caspases, cysteine proteases ----
apoptosis(programmed cell death), necrosis,
and inflammation.
Caspases: maturation of lymphocytes.

Blebbing
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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).

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 Protease assay
Substrate :
casein

Folin & Ciocalteus Phenol, or Folins 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 .

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Fluorokine E Matrix Metalloproteinase
Assay Principle

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REFRENCES

https://www.google.co.in/webhp?sourceid=chrome-
instant&ion=1&espv=2&ie=UTF-8#q=PROTEASE , 26 JULY
2014
http://www.britannica.com/EBchecked/topic/479818/prote
olytic-enzyme
http://www.jbc.org/content/283/45/30433.full
http://en.wikipedia.org/wiki/Endopeptidase
http://en.wikipedia.org/wiki/Exopeptidase
http://www.sigmaaldrich.com/life-science/learning-
center/life-science-video/universal-
protease.html#sthash.6CK
http://www.rndsystems.com/product_detail_objectname_fl
uorokine_e_assay_principle.aspx

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Thank you

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