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Proteases
- 1. PROTEASES Done by : Apurva Fernandes 1
- 2. CONTENTS Peptide bonds Proteolysis Classes of proteases Catalysis Fucntions Specificity Assay Refrences 2
- 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
- 4. Biological strategies: PROTEASES !! also called Peptidases / proteinase found in animals, plants, bacteria, archea and viruses 4
- 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
- 7. 7
- 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
- 11. Catalysis 11
- 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
- 13. Functions.. Blood Coagulation . Thrombin activates fibrinogen resulting in fibrin , causing hemostatis. 13
- 14. Functions .. Digestion Enzymes : Pepsin, chymotrypsin , elastase . 14
- 15. Functions.. Apoptosis Caspases, cysteine proteases ---- apoptosis(programmed cell death), necrosis, and inflammation. Caspases: maturation of lymphocytes. Blebbing 15
- 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
- 18. Fluorokine E Matrix Metalloproteinase Assay Principle 18
- 19. 19
- 20. 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 20
- 21. 21 Thank you
Notas do Editor
- 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.