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For Students in MBBS,BDS,MPT,MSc, in simple language. Dr.Kuldip Singh Sodhi Professor Bio-Chemistry, MMIMS&R MULLANA



2. ENZYMESDR.K.S.SODHI,M. D. PROFESSORBIO-CHEMISTRYMMIMS&R MULLANA AMBALA. 2007 Paul Billiet ODWS 3. The Chemicals of Living Cells The Wellcome Trust 4. HISTORY Of EnzYmESAs early as the late 1700s and early 1800s, the digestion of meat by stomach secretions and the conversion of starch to sugars by plant extracts and saliva were known. However, the mechanism by which this occurred had not been identified. 5. ENZYMOLOGYContribution of Scientists.Definitions.Mode of Action of Enzymes.Factors Influencing Enzyme Activity.Enzyme Inhibition.Regulation of Enzymes.Diagnostic Importance of Enzymes.Therapeutic Use of Enzymes. 6. DEFINITIONSHOLOENZYMES ( APOENZYMES+COENZ.)APOENZYMES; SINGLEPOLYPEPTIDECHAIN,MORE THAN ONECHAIN,MULTI-ENZYME COMPLEX.Co-ENZYMES : Non Protein (VITAMINS)METAL-ACTIVATED ENZYMES.(Zn,Cu,Fe,Mg,K,Ca etc.)ZYMASE: Active without modificationZYMOGENS : Pro Enzymes eg.Trypsinogen 7. ISO-ENZYMES : Physically distinctperform same function. RIBOZYMES: Small ribonuclearparticles. ENDOENZYMES : Produced in thecell. Function inside the cell. EXOENZYMES : Produced inside thecell. Act outside the cell. 8. METALLOENZYMES : Contain metal ionsas essential component. HOUSE KEEPING ENZYMES : Levels ofEnzymes can not be controlled. Alwayspresent in cell. ADAPTIVE ENZYMES : Regulated bygenes. Conc.increase or Decrease. KEY ENZYMES :Regulatory eg HMG-CO.A HYBRID ENZYMES :Produced by geneticfusion. 9. An additional non-protein moleculeCOFACTORSthat is needed bysome enzymes tohelp the reaction Tightly boundcofactors are calledprosthetic groups Cofactors that arebound and releasedeasily are calledcoenzymes Nitrogenase enzyme with Fe, Mo and ADP Many vitamins are cofactorscoenzymes 10. A GOOD TEACHERIS ALWAYS A GOODCATALYST INSTUDENTS LIFE. 11. DISTRIBUTION OF 17 HORSESOLDMAN AND THREE SONS.DISTRIBUTION OF HORSES.ELDER MIDDLE 1/3LITTLE 1/9 12. Notice that without the enzyme it takes a lot more energyfor the reaction to occur. By lowering the activationenergy you speed up the reaction. 13. Energy In Reactions Energy is released orabsorbed wheneverchemical bonds areformed or broken. Because chemicalreactions involvebreaking and formingof bonds, theyinvolve changes inenergy. 14. Enzymes as Biological Catalysts Enzymes areproteins thatincrease the rate ofreaction bylowering the energyof activation They catalyzenearly all thechemical reactionstaking place in thecells of the body Enzymes haveunique three-dimensional shapesthat fit the shapesof reactants(substrates) 15. Thermodynamics 16. The energies of various stages of achemical reaction. Substrates need a largeamount of energy to reach a transition state ,which then decays into products. The enzyme stabilizes the transition state,reducing the energy needed to form products. As all catalysts, enzymes do not alter theposition of the chemical equilibrium of thereaction. Usually, in the presence of an enzyme,the reaction runs in the same direction as itwould without the enzyme, just more quickly. 17. For example, carbonic anhydrasecatalyzes its reaction in either directiondepending on the concentration of itsreactants.(in tissues ; high CO concentration)2inlungs; low CO concentration). 2 18. KineticsEnzyme kinetics is the investigation of how enzymes bind substrates and turn them into products.The enzyme (E) binds a substrate (S) and produces a product (P). 19. In 1902 Victor Henri contributewas to think of enzyme reactions in twostages. In the first, the substrate bindsreversibly to the enzyme, forming theenzyme-substrate complex.This is sometimes called theMichaelis complex. The enzyme then catalyzes thechemical step in the reaction and releasesthe product. 20. In 1902 Victor Henri contributewas to think of enzyme reactions in twostages. In the first, the substrate bindsreversibly to the enzyme, forming theenzyme-substrate complex.This is sometimes called theMichaelis complex. The enzyme then catalyzes thechemical step in the reaction and releasesthe product. 21. Saturation curve for an enzymereaction showing the relation between thesubstrate concentration (S) and rate ( v). 22. Enzyme rates depend on solution conditionsand substrate concentration. Conditions that denature the protein abolishenzyme activity, such as high temperatures,extremes of pH or high salt concentrations.while raising substrate concentration tendsto increase activity. Saturation happensbecause, as substrate concentration increases,more and more of the free enzyme is convertedinto the substrate-bound ES form. 23. At the maximum velocity (Vmax) of theenzyme, all the enzyme active sites arebound to substrate, and the amount of EScomplex is the same as the total amountof enzyme. However, Vmax is only onekinetic constant of enzymes. K m , : is the substrate concentrationrequired for an enzyme to reach one-halfits maximum velocity. Each enzyme has acharacteristic Km for a given substrate.k : is the number of substrate 24. So The efficiency of an enzyme = kcat/Km. This is also called the specificityconstant and incorporates the rateconstants for all steps in the reaction(affinity and catalytic ability). 25. CO-ENZYMES Essential for Biological activity. Low molecular weight, Organic in nature Non protein in nature. .Combine loosely with Enzyme &separatelater. Thermos table. Help in group transfer. Bind to apoenzymes. GTP, NADP, FMN, FAD, Biotin, LipoicAcid, Pyridoxal Phosphate,etc. (Vitamins) Co-enzyme separate from apo-Enz afterreaction. Can be separated by Dialysis. 26. Co-Enzymes can be divided into two groups.A.Oxidoreductases.NADH.NAD PH,FAD.B. Transfer Groups.Thiamine-Hydroxyl group.Pyridoxal phosphate-Amino groupTetrahydrofolate-one CarbonBiotin-Carbon dioxide . 27. Control Points of Gene RegulationTranscription RNA Processing DNA DNARNA Transport 5mRNA RNA Degradationprocess3 ribosome maturemRNAmRNA Translation cap53proteins tailActivity proteinsProteolysisProkaryoticsPost-translationalEukaryotics controlJuang RH (2004) BCbasics 28. Enzyme structureEnzymes are proteinsThey have a globular shapeA complex 3-D structure Human pancreatic amylase 2007 Paul Billiet ODWS 29. STRUCTURE1.MONOMERIC: Single Peptide.2.OLIGOMERIC: Many peptideChains.3.Multienzyme Complex:Fatty Acid SynthaseLDH Complex.Prostaglandin Synthase 30. ENZYMES UNITSKINGARMSTRONG.SOMOGY.REITMAN FRANKEL.SPECTROPHOTOMETRIC.KATAL.INTERNATIONAL UNIT. 31. ENZYMEZS ESTIMATEDFROM:WHOLE BLOOD, SERUM, PLASMA.RED BLOOD CELLS.C.S.F.URINE.SWEAT.SALIVA.SEMEN.AMNIOTIC FLUID.Tears. 32. TISSUESBRAIN,HEART,LIVER,KIDNEY,MUSCLE BRAINMUSCLE HEART KIDNEY LIVERSTOMACHINTESTINE 33. PLASMA ENZYMES FUNCTIONALPLAMSMA ENZYMES. eg. LIPOPROTEIN LIPASE, BLOOD CLOT DISSOLVING ENZYMES etc. NONFUNCTIONAL PLASMA ENZYMES. eg: SGOT, SGPT,AMYLASE,CPK,LDH,LIPASE,ACID -PHOSPHATASE,ALKALINE PHOS., CERULOPLASMIN etc. 34. NATURE OF ENZYMES Soluble,Colloidal, Organic Catalysts Formedby Living Cells ,Specific in action, Protein In Nature ,Inactive at Zero degree centigrade ,Destroyed by moist heat at 100 degree centigrade (Heat Labile), Huge in size, small Active Site, Used for Treatment. 35. DIFFERENCE BIO-CATALYST :Enzymes, protein in nature except ribozymes, More specific, more efficient and slight change in structure alter its action. CATALYST:Inorganic, less sp., less efficient and no change in structure. 36. Compartments of cellDNA, RNA, protein overviewDNARNAMutationsAmino acids,protein structure 37. COMPARTMENTATION MITOCHONDRIA: Enzymes of: E.T.C, TCA Cycle, Beta Oxidation, Urea Cycle, Pyruvate to Acetyle Co-A. CYTOSOL: Glycolysis, HMP Shunt, Fatty Acid Synthesis, Glucogenesis and Glycogenolysis. NUCLEUS: DNA Synthesis, RNA Synthesis and Histones etc. LYSOSOMES : Next Slide 38. FUNCTIONS OF ENZYMES1. Catalyse thousands of reactions. 2.Digestive Enzymes help in igestion. 3.Lysosomal Enzymes destroy in cell. 4.Lysozymes are bacteriocidal, localimmunity(TEARS)4. Detergents5. Textile.6. Leather Industry. 39. What is a Ribozyme?1) Enzyme2) Ribonucleic AcidNOT PROTEIN Sid Altman Tom Cech1989 Nobel PrizeIn Chemistry 40. RIBOZYMESSmall ribonucleic particles.Contain rRNA.Highly substrate specific.Used in Intron splicing,pre RNA to RNA Peptidyl Transferase.Many ribozymes have hair-pin or hammer head shaped active centre &require Divalent Mg++Catalyse reaction on phosphpdiester bonds of other 41. Ribozymes Have followingDrawbacks.Not as efficient as protein catalysts( In RNA there are 4 nucleotides, in amino acid are 20 in number.Act once only in chemical event,protein enzymes are reused several times.Rate of catalytic activity is slower.Synthatic Ribozymes are having better catalytic activity(Cleave infectious Virus) 42. ABZYMESArtificially synthasized catalytic antibodies against Enz. Sub. Complex in transition state of reaction. CATMAB (Catalytic Monoclonal Antibody).Sometimes natural abzymes are found in blood,eg.antivasoactive intestinal peptide autoantibodies.Useful in diseases viz.abzyme against gp120 envelop protein of HIV may prevent virus entry in to the host 43. ANTIENZYMEExtractsof Intestinal Parasites like Ascaris,contain substances called antizymes,which inhibit the action of digestive enzymes pepsin and Trypsin.This is probably the reason why the worms are not digested in the 44. PheRibozyme vs. tRNA folding 45. The Future of Ribozymes In Vitro Molecular Evolution of RNA +High Throughput ScreeningRibozyme-Based Therapies 46. In Clinical Trial...HIV Gene Therapy...Bone Marrow SampleTreat Stem Cells with Retroviral VectorEncodes Gene for anti-HIV Ribozyme Re-Implant Treated Cells 47. ACTIVE SITE OF RIBONUCLEASESItlies in a hydrophobic cleft.7 th Lysine 41 st Lysine on one side and 12 th Histidine and 119Histidine on the opposite side.(URIDYLIC ACID)Peptidyl transferase (chain Elongation)Removal