Enzymes and Enzyme Kinetics
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Transcript of Enzymes and Enzyme Kinetics
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General Concepts on General Concepts on EnzymesEnzymes
&&Enzyme KineticsEnzyme Kinetics
JULIUS P. MARIO, RMT, MSJULIUS P. MARIO, RMT, MS
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HISTORYHISTORY
1878 - 1878 - Willy KUHNEWilly KUHNE coined the term coined the term enzyme (Greek meaning in yeast)enzyme (Greek meaning in yeast)
1907 – 1907 – Edward BUCHNEREdward BUCHNER used used cell-free cell-free extract of yeast to ferment sucrose to extract of yeast to ferment sucrose to ethanol due to ethanol due to zymasezymase
1897 – 1897 – Sir Arthur HARDENSir Arthur HARDEN discovered discovered zymase to be an enzyme mixturezymase to be an enzyme mixture
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1926 – 1926 – James SUMNERJames SUMNER was first to was first to crystallize ureasecrystallize urease
1930 – 1930 – John NORTHROPJohn NORTHROP crystallized crystallized proteolytic enzymes from GITproteolytic enzymes from GIT and and proved proved that they are proteinsthat they are proteins
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Enzyme PropertiesEnzyme Properties Almost are Almost are proteins proteins (ribozymes are (ribozymes are
exceptions)exceptions),, thus have the physicochemical thus have the physicochemical properties of proteinsproperties of proteins
Heat labileHeat labile Water-solubleWater-soluble Can be removed by Can be removed by salting outsalting out process process Made of Made of 16% nitrogen16% nitrogen Deficiency or lack will lead toDeficiency or lack will lead to inborn errors of inborn errors of
metabolismmetabolism Suffix –ase; prefix pro- & suffix –ogen Suffix –ase; prefix pro- & suffix –ogen areare
inactive formsinactive forms
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Enzyme UnitsEnzyme Units Enzyme ActivityEnzyme Activity ( (U/LU/L or or IU/LIU/L))
- amount of enzyme that will - amount of enzyme that will convert 1 micromole convert 1 micromole of substrate per minute per liter of sampleof substrate per minute per liter of sample.(SI .(SI unit is unit is katal katal units = mol/s)units = mol/s)
Specific ActivitySpecific Activity- measure of purity of preparation expressed as - measure of purity of preparation expressed as number of enzymes per mg. of proteinnumber of enzymes per mg. of protein..
Turnover numberTurnover number- - number of substrate converted by a single number of substrate converted by a single enzyme/catalytic siteenzyme/catalytic site; 40,000,000 for catalase, ; 40,000,000 for catalase, 400,000 for CAH & 800 for fumarase.400,000 for CAH & 800 for fumarase.
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Specificity of EnzymesSpecificity of Enzymes
Absolute specificityAbsolute specificity
Bond specificityBond specificity
Group specificityGroup specificity
StereospecificityStereospecificity
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Features of CoenzymesFeatures of Coenzymes Transfer a functional groupTransfer a functional group Essential for enzyme activityEssential for enzyme activity Heat stable low MW organicHeat stable low MW organic substance substance Combines loosely with apoenzymeCombines loosely with apoenzyme Can beCan be separated from apoenzyme via separated from apoenzyme via
dialysisdialysis Can Can bind to two or more substancesbind to two or more substances Derivatives of vitamin B complexDerivatives of vitamin B complex
substancessubstances Involved in oxidoreductasesInvolved in oxidoreductases (e- transport) (e- transport)
and and non-electron transport mechanismsnon-electron transport mechanisms
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MetalloenzymesMetalloenzymesMETALMETAL METALLOENZYMESMETALLOENZYMES
ZincZinc CAH, carboxypeptidases, Alcohol CAH, carboxypeptidases, Alcohol dehydrogenasedehydrogenase
MagnesiumMagnesium Hexokinase, PFK, enolase, G6PaseHexokinase, PFK, enolase, G6Pase
ManganeseManganese Phosphoglucomutase, hexokinase, enolase, Phosphoglucomutase, hexokinase, enolase, glycosyl transferaseglycosyl transferase
CopperCopper Tyrosinase, Cytochrome oxidase, lysyl oxidase, Tyrosinase, Cytochrome oxidase, lysyl oxidase, SODSOD
IronIron Cytochrome oxidase, catalase, peroxidase, Cytochrome oxidase, catalase, peroxidase, XODXOD
CalciumCalcium Lecithinase, lipaseLecithinase, lipase
MolybdenumMolybdenum XODXOD
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ProteasesProteases Serine proteases – Trypsin, Chymotrypsin, Serine proteases – Trypsin, Chymotrypsin,
clotting factorsclotting factors
Cysteine proteases – PapainCysteine proteases – Papain
Aspartyl proteases – Renin, retroviral proteasesAspartyl proteases – Renin, retroviral proteases
Carboxyl proteases – PepsinCarboxyl proteases – Pepsin
Metalloproteases – CarboxypeptidasesMetalloproteases – Carboxypeptidases
Protease inhibitors – ACE inhibitor (Captopril);Protease inhibitors – ACE inhibitor (Captopril); HIV protease inhibitor HIV protease inhibitor
(Retonavir)(Retonavir)
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Active Site/Active CenterActive Site/Active Center EnzymesEnzymes Catalytic groupsCatalytic groups ChymotrypsinChymotrypsin His (57), Asp(102), Ser (195)His (57), Asp(102), Ser (195) TrypsinTrypsin Ser, HisSer, His ThrombinThrombin Ser, HisSer, His PhosphoglucomutasePhosphoglucomutase SerSer ALPALP SerSer AcCHEAcCHE SerSer CAHCAH CysCys HexokinaseHexokinase HisHis CarboxypeptidaseCarboxypeptidase His, Arg, TyrHis, Arg, Tyr ALSALS LysLys
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Reactions & Energy Reactions & Energy UtilizationUtilization
Exergonic/Exothermic reactionsExergonic/Exothermic reactions
Isothermic reactionsIsothermic reactions
Endergonic/Endothermic Endergonic/Endothermic reactionsreactions
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Derivation of KmDerivation of Km k1 k3k1 k3 E + E + SS ES ES E + E + PP k2k2
KmKm = k2 + k3/k1 = ½ Vmax = k2 + k3/k1 = ½ Vmax
Km value Km value 1/ enzyme affinity for substrate 1/ enzyme affinity for substrate
MM equation: v = Vmax [S] MM equation: v = Vmax [S] // Km + S Km + S
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Factors that Affect Factors that Affect Enzymatic ActivityEnzymatic Activity
Enzyme concentrationEnzyme concentration Substrate concentrationSubstrate concentration Product concentrationProduct concentration TemperatureTemperature [H[H++]] ActivatorsActivators InhibitorsInhibitors Repressors/derepressorsRepressors/derepressors
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Competitive InhibitorsCompetitive InhibitorsDRUG ENZYME INHIBITED CLINICAL USEDRUG ENZYME INHIBITED CLINICAL USE
AllopurinolAllopurinol XOD XOD Gout Gout DicoumarolDicoumarol VitK epoxide reductase AnticoagulantVitK epoxide reductase Anticoagulant
PenicillinPenicillin Transpeptidase BacteriaTranspeptidase Bacteria Sulphonamide Pteroid synthetase BacteriaSulphonamide Pteroid synthetase Bacteria Trimethoprim FH2-reductase BacteriaTrimethoprim FH2-reductase Bacteria Pyrimethamine FH2-reductase MalariaPyrimethamine FH2-reductase Malaria Methotrexate FH2-reductase CancerMethotrexate FH2-reductase Cancer
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Competitive InhibitorsCompetitive InhibitorsDRUG ENZYME CLINICAL DRUG ENZYME CLINICAL INHIBITED USEINHIBITED USE
6-mercaptopurine adenylosuccinate synthetase cancer 6-mercaptopurine adenylosuccinate synthetase cancer
5-fluorouracil thymidylate synthetase cancer5-fluorouracil thymidylate synthetase cancer
Azaserine phosphoribosylamidotransferase cancerAzaserine phosphoribosylamidotransferase cancer
Cytosine arabinoside DNA polymerase cancerCytosine arabinoside DNA polymerase cancer
Acyclovir DNA polymerase virusAcyclovir DNA polymerase virus
Neostigmine AChE myastheniaNeostigmine AChE myasthenia
Alpha methyl dopa dopa decarboxylase hypertensionAlpha methyl dopa dopa decarboxylase hypertension
Lovastatin HMG CoA reductase lowering of cholesterolLovastatin HMG CoA reductase lowering of cholesterol
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Noncompetitive Noncompetitive (Irreversible) Inhibitors(Irreversible) Inhibitors
CyanideCyanide - inhibits cytochrome oxidase - inhibits cytochrome oxidase FluorideFluoride - removes Mg++ and inhibits - removes Mg++ and inhibits
enolase & consequently glycolysisenolase & consequently glycolysis IodoacetateIodoacetate - inhibits SH-containing - inhibits SH-containing
enzymesenzymes BALBAL - also dimercaprol; antidote for - also dimercaprol; antidote for
heavy metals (SH reactive)heavy metals (SH reactive)
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Thank you!!!Thank you!!!