Enzyme-1 – Structure and Isoenzymes lecture NO : 1 st MBBs

1. Enzyme-1 – Structure and Isoenzymes lecture NO : 1st MBBs Dr Muhammad Ramzan

2. Enzyme (E) – the definition • A protein that catalyzes chemical reactions of other substances • without itself being consumed or altered upon the: • Completion of the reactions. • Enzymes lower the activation energy • Enzymes are produced by the livingorganisms • www.biology- online.gov www.medicaldictionary.com

3. Enzymes (E) – the backgroundIntra/extra cellular • All Es are proteins except a few catalyticRNAs- Ribozymes + CK • Defect or deficiency of an E produces diseases called "inborn error of metabolism.“ like Lactose Intolerance • Human body contains about 10,000 different enzymes • Some Es workwithin the cell ( RNA polymerase) and others outside the cells like digestive enzymes (Pepsin and Trypsin) • The substance acted upon by an E is called a Substrate – S • www.en.wikepedia.org - www.medicaldictionary.com

4. Lactose Intolerance

5. Biological significance of Enzymes Reduces activation energy • At body temperature, very few biochemical reactions proceed at a significant ratewithout the presence of an E 1 • Es lower the activationenergy of a catalyzed reaction, thus • increasing the rate of the reaction 2 • They do so in a step by step, highlyefficient and safe manner 3 • Activation energy is the minimumenergy, required to start and complete a reaction • www.medicaldictionary.com

6. Enzyme Lowers the Activation Energy

7. Parts of Enzymes(E) - 2 parts body and active site • Enzymes are commonly proteins and variable in size • Each enzyme has a specific area to bind the substrate by the noncovalently/temporary bonds • Essentiallyenzymes have 2 parts: • Body of enzyme and • Active site • www.tutorvista.com - www.medicaldictionary.com

8. Structure of enzyme – 2 partsBody and active site

9. Body of enzyme - 1Globular and variable in size • Es are globular proteins and their AA content vary from 62 to 2500 AA residues a (Fatty acid Synthase. FAS ) • Es are larger than S and some are grouped together to formenzyme complex like FAS/LDH - 6/4 units • Es are long linear chains of AAs but their activities are due to their 3D Tertiary structures • Apozyme is inactive enzyme with protein portion only • Holozyme is an active enzyme = proteinand cofactor/coenzyme • www.en.wikipedia.org

10. Apo and Holoenzymes

11. Active site of enzyme 2 Produced by protein folding • Active sites are the hollows/cracks/ pockets on the surface of E and are produced by protein folding • Active site assumes 3D structure • Active site has 2- 4 AAs that are involveddirectly in Substrate binding and catalysis • S binding and catalysis is also due to the electrical and chemical properties of side chains of Amino acids at: • Active site(+ and – ive)

12. Active site – Mechanism for S binding • Substrate binding is temporary by non covalent bonds • It is relieved after the completion of the catalysis • The binding/catalysis is due to the Amino Acyl or functional groups of the AAs involved • Both, S and AAs of the active site have the compatiblefunctional groups, sequence of AAs and shape • The usual analogy for this is a key fitting exactly into the lock

13. Parts of active site

14. Cofactor - Inorganic substancesA substance other than substrate • Cofactors are non proteininorganic substances, needed to activate the Es to carry out a specific catalysis • Bind to active site of E like: Zinc to Carbonic anhydrase • Fe2+/Fe3 + Cytochromes (ETC) / and HB(Fe3) • K+ and Mg++ to PyruvatePhosphokinase • They are called helper molecules as they assist the enzymesto carry out catalysis • www.biology- on line.org – www. www.wikepedia - www.biologydictionary.net

15. Co enzymes • Coenzymes are organiccompounds that help the enzyme in catalyzing a chemical reaction • They are usuallywater soluble vitamins like : NAD,NADH and • FAD for the transfer of O2 and H2 • Acetyl COA for the transfer of functional groups like Acyl group • They bind to the active site of enzyme for activation and act as carrier of intermediate products • www.biology online.org

16. Co enzymes and Cofactors

17. Co factors and co enzymes

18. Enzymes - Principle of working E identifies and binds S • EnzymeidentifiesSubstrate and binds it to E active site. • Enzyme holds the substrate in a particularGeometric position and forms enzyme substrate complex (ESC) • This binding is temporary and non covalent • ESC gets activated and catalyzed to its products • Enzyme lowers the activation energy

19. Enzymes – Principal of working cont.E controls the direction of reaction • Both, the enzyme and products are released upon completion of the reaction and enzyme is available for Reuse • The enzymedoes not control the direction of the reaction. • It is controlled by the cell itself • Cell ↑the rate of forward and reverse reactions proportionallydepending upon the bodyneeds (Regulation) • www.wikibooks.org

20. Working of enzymes – the principals

21. Enzymes – Quantity and location/TypesTypes/ within and outside the cell • Most enzymes are produced in tiny quantities and catalyze reactions within the cells /Intra cellular like: • DNA/RNA Polymerase and HMG CoA Synthase/Catalase • Extra cellular: Digestive enzymes are produced in large quantities and act outside the cells/ lumen of the GIT • They cleave the dietarymacromolecules like TG,Glycogen and Nucleic acids into smaller ones/ monomers • www.wikepedia.org

22. Allosteric or Additional sites Allosteric enzymes • Enzymes can also have sites other than active site that bind cofactors, which are needed for catalysis • These sites also serve to bind products and S for catalysis • These are Allostericsites and enzymes are Allosteric ones • Serve to regulate the enzymeactivity via negative feed back through activators/inhibitors

23. Allosteric enzymes

24. Denaturation of enzymes – heat and pH • Most enzymes can be denatured - unfolded and inactivated • by heating or chemicaldenaturants / change of pH 1 • It disrupts the 3 structureof the protein (E) and 2 • Results in loss of function 3 • Denaturation of E may be reversible or irreversible depending upon the cause and damage to the enzyme

25. Denaturation of enzyme

26. Isoenzymes/ Isozymes – the Definitiondifference in sequence of AAs • Isoenzymes are generally the Multiple forms of the enzymes that differ in AA sequencebutcatalyze the : • Same chemicalreaction. • These include the Isoenzymes of the : • Lactic Dehyrogenase (LDH) ,Creatine Kinase (CK) and Alkaline Phosphatase • www.biology- online.org

27. Isoenzymes - Background • Isoenzymes display different electrophoreticmobility, regulatory and immunological properties • Isozymes are the result of gene duplication • These genes expresspolypeptideschains that producedifferentsubunits of the Isoenzymes • Like CK has 2 genes (M+B) that express 2 Polypeptidesubunits – M and B • These produce CK1,CK2 and CK3 • www.biology-online.org

28. Isoenzymes – Basis for elevated levels (CK) • Differentorgans of the body contain the characteristicproportions of the differentIsoenzymes • The pattern /conc. of Isozymes in the plasma may identify the site of tissue damage like CK has 3 forms • These Isoenzymes is present in 3 different tissues • The damage to these tissues releases  amount of respective CK Isoenzymes in circulation(Heart, brain and muscles)

29. Isoenzymes of Creatine Kinase

30. Isoenzymes of LDH

31. Creatine Kinase – Clinical importance • Level of Creatine Kinase CK (CKMB) is commonly determined for the diagnosis of Myocardial infarction – MI • These Isoenzymes are particularly useful when ECG is difficult to interpret especially when there have been : • Previous episodes of MI • Same is true for Cardiac Troponin T and I