enzymes and enzyme kinetics 2012

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Enzymes and enzyme kinetics 2012

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  • 1. ENZYMES

2. OBJECTIVESAt the end of the lecture, the student should beable to: 1. Define the following:a. Enzymesb. Apoenzymec. Coenzymed. Holoenzymee. Metalloenzymef. Regulatory enzymeg. Active site of the enzymeh. Allosteric site of the enzymei. Substrate 2. Discuss the helpers (cofactors) of enzymes. 3. OBJECTIVES3. Enumerate the six major classes of enzymes.4. Discuss the characteristics of enzymes.5. Explain the models of enzyme-substrate complex.6. Explain enzyme kinetics. a. Factors that affect enzyme activity or reaction velocity.b. Ways of expressing enzyme activity.7. Discuss the operation and plots used to illustrate enzyme kinetics.a. Michaelis-Menten kineticsb. Lineweaver-Burke Double Reciprocal Plotc. Michaelis constant and its significanced. Kinetic order of reactions 4. OBJECTIVES8. Discuss enzyme inhibition and its effect on reaction velocity.a. Reversibleb. Irreversible9. Discuss the different ways of regulating enzyme activity.10. Explain the factors affecting enzyme activity.11. Elucidate uses and clinical application of enzymes. 5. ENZYMESSpecialized protein catalyststhat accelerate chemicalreactions 6. DEFINITION OF TERMSAPOENZYME APOENZYMEAPOENZYMEProteinpart +++Prosthetic Cofactor MetalCoenzymegroup(Nonproteinionpart) HOLOENZYME 7. ENZYME COFACTORSChemicalVitaminA. CoenzymeEnzymeGroupsPrecursor Transferred ThiaminePyruvate dehydrogenase,PyrophosphateIsocitrate dehydrogenase, - AldehydesThiamine(TPP)ketoglutarate dehydrogenase, Transketolase, -Ketoacid (Vit B1) dehydrogenase Flavin AdenineSuccinate dehydrogenase, - RiboflavinDinucleotide Ketoglutarate dehydrogenase, Electrons (Vit B2)(FAD)Pyruvate dehydrogenase, Nitric oxide synthase Nicotinamide Lactate dehydrogenase; Hydride ionNicotinic acid Adenine Other dehydrogenases (:H-)(Niacin; B3)Dinucleotide (NAD) Glycogen phosphorylase, Pyridoxine Pyridoxal -ALA synthase, HistidineAmino groups(Vit B6)Phosphate (PLP)decardoxylase, Alanine aminotransferase Lipoate Pyruvate dehydrogenase Electrons and Not required-Ketoglutarate dehydrogenaseacyl groupsin diet 8. ENZYME COFACTORS ChemicalVitaminA. CoenzymeEnzyme GroupsPrecursorTransferredCoenzyme A Acetyl CoAPantothenic (CoASH) carboxylaseAcyl groupsacid & other compounds Pyruvatecarboxylase,CO2 Acetyl CoABiocytinBiotincarboxylase, Propionyl CoAcarboxylase5- Methylmalonyl H atoms andVit B12deoxycobalamin mutase alkyl groups Thmidylate One-carbonFolic acidTetrahydrofolalatesynthasegroups 9. CLASSES OF COENZYMESCLASS EXAMPLESTPPActivation-TransferCoenzyme ACoenzymes BiotinPLPOxidation-ReductionNAD+Coenzymes FADVit E, Vit CMarks Medical Biochemistry, 3rd ed. 10. ENZYME COFACTORS: COENZYME A OCH3 Pantothenic acidIIH I C-CH2-CH2-N-C-CC-CH2O I II I I NHO OH CH3 I O = P O- CH2 I1. Pantothenic acid-derived, O I O co-factor of severalNH2 II CH2 IN enzymes like acetyl CoA I NHO = P O- carboxylase.SHN IN2. Takes part in reactions ofH O O the CAC, FA synthesis and oxidation, acylations andHHH cholesterol synthesis. OH O Activesulfhydryl Igroup that formO = P O- I thioesters with O- acyl groups 11. ENZYME COFACTORS:COENZYME A-Ketoglutarate Dehydrogenase ComplexCoenzymes: COO-COOH1. TPP2. Lipoic AcidPyruvate decarboxylase ||3. FADDihydrolipoyl transacetylase CH2CH2 4. NAD+ Dihydrolipoyl dehydrogenase|| 5. CoASH CH2CH2 || C=OC=O CO2 || CoASHCOO - NAD+NADH S ~ CoA + H+-Ketoglutarate (C5) G0 = - 8.0 kcal Succinyl CoA (C4) 12. ENZYME COFACTORS: COENZYME APyruvate Dehydrogenase Complex COO- TPP Pyruvate decarboxylaseS ~ CoALipoate Dihydrolipoyl transacetylase |FAD Dihydrolipoyl dehydrogenase | C=OC=O || CH3CH3Pyruvate CoASHNADH+ + CO2 Acetyl CoA(C3) NAD+H+(C2) G0 = - 8.0 kcal/mole 13. ENZYME COFACTORS: NAD & NADPPyruvate COO-Lactate I 1 COO-KetoC=O + H+ DissociatesI group Ias H+2H-O-C-HCH3 Functional INicotinamide adenine dinucleotide (NAD+)3 CH3H OgroupO CII II C NH2CC NH2 Nicotinamide ringLactate+dehydrogenase NN1 I -O O CH2 O RP HH Ribose-OOHH ringOH OHNH2 N 6 O75 1NO8Adenine2 4 AMP provides additional 9 3 2 ring AMP P N binding interactions that 5N-OO CH2 Oinduce conformational4 1changes in the enzymeHH H2 H 3NADP+ contains a P3OH2 on this 2-hydroxyl OH 14. ENZYME COFACTOR: NAD+Lactate COO-COO- dehydrogenase | IHO - C H C=O | ICH3 CH3L-LactateNAD+ NADH+ + H+ Pyruvate 15. COENZYME: BIOTIN OII H-NN-H HIIIIIIIIIIIIIII IIIIIIIIIIIIIII HR SBiotin H Protein portion of enzyme:1. One of the B complex Acetyl CoA carboxylase vitamins. Propionyl CoA carboxylase 2. A cofactor of such Pyruvate carboxylaseenzymes as acetylNCCoA carboxylase,HII propionyl CoA O Lysyl O Ocarboxylase andresidueII IINH pyruvate carboxylase.H-N N-H=O3. It is a carrier of activatedCO2H IIIIIIIIIIIIIII IIIIIIIIIIHCO2, hence involved inattachmentBiotincarboxylation reactions.site SH 16. COENZYME: BIOTINCOO- BIOTIN CO2 |COO-ATP ADP + PiC= OI |C =OCH2I |CH3 COO- PyruvatePyruvatecarboxylaseOxaloacetate Gluconeogenesis 17. COENZYME:THIAMINE PYROPHOSPHATE Reactivecarbon atom:carrier of aldehydeDissociable groupsH proton C-C-OHCoenzymeHHH bindingC NN S NNH2 siteHNH CC NC CH2 N S O- O- C H3C NCNH2 C - CH2 CH2 O P O P O- IIO IICH O3H3C Thiamine PyrophosphateAMP (TPP; Vit B1-derived) 18. PYRUVATE (C3)Role of TPP in theNAD PyruvateOxidative Decarbo-TPPxylation Reactions dehydrogenaseNADH + H+of CAC ACETYL-CoACO2 (C2) Citrate synthase (C6) OXALOACETATE H2OCITRATE (C6) Malate NADH + H+ Aconitase dehydrogenase H2ONAD(C4) MALATEISOCITRATE (C6)NAD Isocitrate Fumarase TPPH2OEdehydrogenase ATP T NADH + H+ CO2 (C4) FUMARATE Oxid. CPhospho a KETOGLUTARATE (C5)FADH2 NADSuccinate TPP a - ketoglutaratedehydrogenase FADH2O O2 NADH + H+CO2 dehydrogenase (ATP)(C4) SUCCINATE GTP ADP + Pi SUCCINYL CoA (C4)Mg+CoA Succinyl CoA thiokinase 19. COENZYME: FAD H3C NNH2 NH Isoalloxacin ring H3CN NOCH2 From ATPH3C IH-C-OHIH-C-OHNH2Ribitol IH-C-OH ON NOI IIIICH2-O-P O P O-CH2 NP N I OOO OH OHFlavin Adenine Dinucleotide (FAD)A riboflavin (Vit B3)-derived coenzyme of several dehydrogenases involved in oxidation-reduction reactions. 20. COENZYME: FADROLE OF FAD IN THE CITRIC ACID CYCLECOOH SuccinateH -COOH|dehydrogenase |CH2 HC|||CH2CH| |COOH H -COOH FADFADH2Succinate (C4) Fumarate (C4) 21. COENZYME: FADNH2ROLE OF FAD AND FMN IN NH2 + IH2N = C NITRIC OXIDE (NO) SYNTHESIS I I C=ONHINADPH2 NADP+ NHICH2+ O2+ H2O IICH2CH2IICH2CH2INO ++ ICH2H3N C H Nitric oxide synthase+ IIH3N C HCOO-FAD, FMN, Heme ITetrahydrobiopterinCOO- ArginineCitrulline 22. COENZYME:PLPReactive aldehyde groupinvolved in the transferO of amino groups. O C HH -O PO-CHOH 32+NCH3H Pyridoxal Phosphate A Vit. B6-derived coenzyme involved incarbohydrate, amino acid and neurotransmitter synthesis. 23. COENZYME: PLPROLE OF PLP IN CARBOHYDRATE METABOLISM: GLYCOGENOLYSIS HOOHOOGlycogen HOOHOOHchain OHOOHO OH OH HOOH H OHHOHHOHHOHHOH Pi PLPGlygogenphosphorylase HOO HOOHOOHO OHOHOHO-PO3 = + OH OOH HOOHHOH OHHOH HOHH OHGlucose 1-PRemaining glycogen 24. COENZYME: PLPROLE OF PLP AS A COENZYME IN AMINO ACID METABOLISM: HEME SYNTHESISGlycine + Succinyl CoA-Aminolevulenate synthasePLP-Aminolevulenic acid(ALA)several reactionsHeme(Fe protoporphyrin IX) 25. COENZYME: PLPROLE OF PLP IN AMINO ACID METABOLISM: HISTAMINE SYNTHESISH|CH2 C COO-|NH3Histidine HistidinePLP decarboxylaseCO2 CH2 CH2 NH3 HISTAMINE 26. COENZYME: PLPROLE OF PLP IN AMINO ACID METABOLISM:TRANSAMINATIONCOO- COO-| Alanine |CH2aminotransferase CH2|NH3+ (transaminase)|CH2 | CH2| O+CH3 C C COO- +C=O || | |H C NH3+|PLPCH3 C COO-H|COO-Pyruvate COO- Alanine-Ketoglutarate Glutamate 27. ENZYME COFACTORS CofactorEnzymeB. Inorganic (Metal ions or iron- sulfur clusters) Zn+2Carbonic anhydrase, Alcohol dehydroge- nase, Carboxypeptidases A & B Cu+2Cytochrome oxidase Mn+2Arginase, Ribonucleotide reductase Mg+2Hexokinase, Pyruvate kinase, Glucose 6-phosphatase Ni+2UreaseMo Nitrate reductaseSe Glutathione peroxidase Mn+2Superoxide dismutaseK+ Propionyl CoA carboxylase 28. ENZYME COFACTORS: Mg+2OO ||||C1 - H C1 - H ||H - C2 - OH Hexokinase H - C2 - OH | OH - C3 - HGlucokinase |OH - C3 - H ||H - C4 - OH H - C4 - OH ||H - C5 - OH |Mg+2 H - C5 - OH|H - C6 OH ADP H - C6 - O P |ATP |H + PiHGlucose Glucose 6-PO4 29. GLYCOLYSIS ENZYME COFACTORS: K+O||C 1 O- Pyruvate kinase |COO-C2 O ~ P| |C2 = OH C3 K+ | |CH3H ADP ATPPhosphoenol Pyruvate Pyruvate (PEP) G0 = - 6.1 kcal/mole 30. ENZYME COFACTORS: Zn+Carbonic anhydraseCO2 + H2OH2CO3Zn+2 31. METALLOENZYMESEnzymes that requirea metal in their composition 32. SUBSTRATEThe molecule acted upon by the enzyme to form aproduct 33. ATP AS A CO-SUBSTRATE O O ||||C1 - H C1 - HHexokinase/ ||GlucokinaseH - C2 - OH H - C2 - OH ||OH - C3 - HOH - C3 - H||Mg+2 H - C4 - OH H - C4 - OH || H - C5 - OH ATP ADP H - C5 - OH ||H - C6 - O P H - C6 OH G0 = - 4.0 kcal/mole ||H H GlucoseGlucose 6-Phosphate 34. ACTIVE SITE OF THE ENZYME 35. LYSOZOME: ACTIVE SITE 36. CHYMOTRYPSIN:ACTIVE SITE His 57 Ser 195 37. ALLOSTERIC SITESubstrateSubstrate sitesEnzymeAllosteric site 38. REGULATORY ENZYMEThe enzyme that catalyzes therate-limiting or committedstep of a metabolic pathway. 39. REGULATORY ENZYME Phosphofructokinase I HH|| H - C1 - OHH - C1 - O - P|ATPADP + Pi | C2 = O C2 = O||OH - C3 - HOH - C3 - H|| H - C4 - OHH - C4 - OH||Phosphofructokinase I H - C5 - OHH - C5 - OH|+ - | H - C6 - O PATPH - C6 - O P|AMP | Citrate H F 2,6 bisPO4 + HHFructose 6-Fructose 1,6-phosphateGlycolysisbisphosphate 40. REGULATORY ENZYME Acetyl CoA CarboxylaseO Citrate|| Insulin CH3 C S CoA High CHOCO2 ACETYL CoA Low Fat (HCO3-) High Prot.ATP +Acetyl CoA carboxylase ADP + Pi - H2O Malonyl CoA Palmitoyl CoA EpinephrineO O Glucagon|| High Fat C CH2 C S CoA Fasting //OMALONYL CoADe Novo Synthesis of Fatty Acids 41. REGULATORY ENZYME HMG Coa Reductase Insulin, T3 Cholesterol Glucagon Glucocorticoids Bile acidsStatins Mevalonate O II+ - C1OO--O C| I HMG CoA reductase C 2H 2 CH2| I HO C3 CH3OH C CH2| IC 4H 2 C S Co

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