Biochemistry Lecture Notes

EE Y M EE This is not a difficult foreign language… N It’s just a Z Biochemistry Lecture  Biochemistry Lecture ENZYM E Introduction into the World of Catalysis and BIOCHEMISTRY  Biochemistry Review ENZYM E Intro...

EE Y M EE This is not a difficult foreign language… N It’s just a Z Biochemistry Lecture  Biochemistry Lecture ENZYM E Introduction into the World of Catalysis and BIOCHEMISTRY  Biochemistry Review ENZYM E Introduction into the World of Catalysis and BIOCHEMISTRY  Lecture outline Part 1: General Part 2: Enzyme Concepts Kinetics and Inhibition Enzyme characteristics Concept of rates, and mechanism of action reaction rates and kinetics Enzyme structure Michaelis-Menten Curve Enzyme nomenclature and classification Lineweaver-Burk Graph Enzyme regulation Enzyme Inhibition  Part 1 General Concepts  At the end of your study, you are expected to.... Define, name enzymes Discuss properties and structure of enzymes Discuss and explain mechanisms of enzyme catalysis Discuss and explain enzyme activity control  Biomedical importance Life is not possible without enzymes Enzyme deficiency may lead to diseases Enzymes are therapeutically targeted by some drugs May be used for diagnostic markers Utilized as analytical reagents in research  What are enzymes? Proteins which accelerate chemical reactions Some are RNA (ribozymes) Regulate metabolic reaction rates Biological catalyst  Mechanism of enzyme action Enzyme Uncatalyzed Energy of Energy of Activation Activation lowered by Reactant Reactant enzyme Uncatalyzed reaction pathway Enzyme-catalyzed reaction pathway  Mechanism of enzyme action Enzyme Uncatalyzed Energy of Energy of Activation Activation lowered by Reactant Reactant enzyme Uncatalyzed reaction pathway Enzyme-catalyzed reaction pathway BOTTOMLINE Enzymes lower activation energy necessary to transform a reactant into a product. Enzyme-catalyzed reaction pathway has a smaller energy barrier (activation energy) to overcome.  Mechanism of enzyme action Enzyme bind substrate at active site/ catalytic site Enzyme Active site fits shape of substrate Allosteric Active E+S ES E+ P site site Association between enzyme and substrate is temporary Substrate Allosteric site : any site other than active site Models of enzyme-substrate binding Lock-and-Key Active site complements substrate precisely Explains how enzyme specificity for a particular substrate Enzyme Substrate Models of enzyme-substrate binding Lock-and-Key Active site complements substrate precisely Explains how enzyme specificity for a particular substrate Enzyme Substrate Complex Models of enzyme-substrate binding Lock-and-Key Active site complements substrate precisely Explains how enzyme specificity for a particular substrate Enzyme Transition state Complex Models of enzyme-substrate binding Lock-and-Key Active site complements substrate precisely Explains how enzyme specificity for a particular substrate Enzyme Product Models of enzyme-substrate binding Active site is NOT a Induced Fit completely rigid fit for the substrate ++ Active site will undergo - a conformational change when exposed to a substrate + - Explains enzymes broad specificity Enzyme Substrate Models of enzyme-substrate binding Active site is NOT a Induced Fit completely rigid fit for the substrate Active site will undergo + - a conformational change when exposed to a substrate + - Explains enzymes broad specificity Enzyme Substrate Complex Models of enzyme-substrate binding Active site is NOT a Induced Fit completely rigid fit for the substrate Active site will undergo + - a conformational change when exposed to a substrate + - Explains enzymes broad specificity Enzyme Substrate Complex Models of enzyme-substrate binding Active site is NOT a Induced Fit completely rigid fit for the substrate Active site will undergo + - a conformational change when exposed to a substrate + - Explains enzymes broad specificity Enzyme Transition state Complex Models of enzyme-substrate binding Active site is NOT a Induced Fit completely rigid fit for the substrate Active site will undergo + - a conformational change when exposed to a substrate - + Explains enzymes broad specificity Enzyme Product Complex Models of enzyme-substrate binding Active site is NOT a Induced Fit completely rigid fit for the substrate - Active site will undergo + a conformational change when exposed to a substrate - + Explains enzymes broad specificity Enzyme Product Structure of enzyme ENZYMES Complex or Holoenzymes Simple  Structure of enzyme ENZYMES Complex or Holoenzymes Simple Apoenzyme Cofactor  Structure of enzyme ENZYMES Complex or Holoenzymes Simple Apoenzyme Cofactor Prosthetic group Coenzyme Usually contain Large organic small inorganic nonprotein molecule molecule or atom; Loosely bound to Tightly bound to apoenzyme apoenzyme  Structure of enzyme Holoenzyme is an active enzyme with its non protein component. Enzyme without its non protein moiety is termed as apoenzyme and it is inactive  Structure of enzyme A cofactor is a non-protein chemical compound that is bound (either tightly or loosely) to an enzyme and is required for catalysis. Types of Cofactors: Coenzymes Prosthetic groups  Structure of enzyme Coenzyme: Non-protein component, loosely bound to apoenzyme by non-covalent bond. Examples : vitamins or compound derived from vitamins. Prosthetic group: Non-protein component, tightly bound to apoenzyme by covalent bonds How are enzymes named? Substrate + “ase” Reaction + “ase” Based on name of substrate Most enzymes named after their substrate "-ase" suffix added to the substrate name Examples: Lactase (acts on lactose), Lipase (acts on lipids) Based on name of substrate substrate products Based on type of reaction Many enzymes named after the type of reaction catalyzed "-ase" suffix added to the substrate name Examples: succinate dehydrogenase, pyruvate decarboxylase Based on type of reaction Based on type of reaction  EXCEPTIONS!!!!! How are enzymes named? Proteolytic enzymes’ suffix is ‘in’ Chymotrypsin Pepsin Restriction endonuclease EcoR1 Mst2  How are enzymes classified? Class Type of reaction Oxidoreductase Transfer of electrons Transferase Group transfer reactions Hydrolase Hydrolytic reactions Lyase Addition/ removal of groups Isomerase Structural rearrangement Ligase Condensation reaction coupled to ATP hydrolysis  Oxidoreductases Transfer electrons between molecules "Oxido" refers to loss of electron "Reductase" refers to reduction (gaining electrons) May use NAD or FADH as their cofactor/electron acceptor Oxidoreductases  Transferases Transfer functional groups between molecules Functional groups are like molecular building blocks Examples: Kinase (transfers phosphate groups), Glutamate transaminase (transfers amino groups) Transferases Transferases  Hydrolyases Break down molecules using water "Hydro" refers to water "Lyase" refers to breaking bonds Examples: Lactase  Hydrolyases substrate products  Lyases Break down molecules by removing functional groups without water Different from hydrolases: Hydrolases use water, lyases don't Examples: Pyruvate decarboxylase (removes CO2) Based on type of reaction  Isomerases Rearrange atoms within a molecule Create isomers: molecules with same formula, different structure Examples: Hexose isomerase (glycolysis enzyme) Isomerases  Ligases Join two separate molecules together Use energy from ATP (adenosine triphosphate) Different from transferases & lyases: ligases form new bonds, while transferases move groups and lyases break bonds without water Ligases  Enzyme regulation Irreversible covalent modification/ Zymogen cleavage Allosteric regulation Reversible covalent modification Genetic control  Irreversible covalent modification/ Zymogen cleavage Proteolytic enzymes are stored as zymogens or proenzymes. Zymogen = inactive Zymogen activation is a cleavage process  Irreversible covalent Activation of chymotrypsinogen modification/ by proteolysisZymogen cleavage Activation of chymotrypsinogen Irreversible covalent by proteolysis modification/ Zymogen cleavage Activation of chymotrypsinogen Irreversible covalent by proteolysis modification/ Zymogen cleavage  Irreversible covalent Activation of chymotrypsinogen modification/ by proteolysisZymogen cleavage  Irreversible covalent modification/ Zymogen cleavage  – Precursors fold in 3 dimensions – Later activated by enzyme-catalyzed cleavage (hydrolysis) of 1 or Irreversible covalent more specific peptide bonds ZYMOGENS (or proenzymes): inactive precursors modification/ Zymogen cleavage zymogen activation: cleavage/activation process Examples: 1) mammalian digestive enzymes Gasctric and pancreatic zymogens  Allosteric regulation Regulation of activities of an enzyme caused by reversible noncovalent binding of regulators at the site other than the active site Allosteric enzyme can oscillate from active form to inactive form Allosteric feedback inhibition/ activation of enzyme in a biochemical pathway Allosteric regulation Enzyme Allosteric Active site site Substrate Allosteric regulation Enzyme Allosteric regulation Enzyme Allosteric regulation Enzyme Products Allosteric regulation Enzyme Products Allosteric regulation Enzyme Products Allosteric regulation Enzyme Products Allosteric regulation Enzyme Allosterically inhibited Products Allosteric regulation Enzyme Allosterically inhibited Products Allosteric regulation Enzyme Allosterically inhibited Products Allosteric regulation Enzyme Allosterically inhibited Products Allosteric regulation Enzyme Allosterical inhibition is reversed Allosteric regulation Enzyme Allosteric regulation Enzyme Allosteric regulation Enzyme  Product of a pathway controls the rate of its own synthesis by inhibiting an early step (usually the first “committed” step (unique to the pathway) Allosteric regulation  Allosteric regulation Final product accumulates in abundance Excess final product binds to an allosteric site on the first enzyme in the series of reactions Inhibited enzyme results to less product produced Immediate response Inhibition is reversible  Covalent modification Modification of catalytic or other properties of proteins by covalent attachment of a modifying group (phosphate) Enzymes can cycle between active and inactive states by chemical modification Slower and longer-lasting effects Exemplified by glycogen phosphorylase  Covalent modification Glucose glycogen Glycogenesis Glycogenolysis phosphorylase Glycogen  Control of glycogen phosphorylase activity GLUCAGON P 2 ATP 2 ADP kinase phosphatase 2 Pi 2 H2O P Inactive INSULIN Active  Covalent modification Reversible covalent modification of enzyme activity via phosphorylation/dephosphorylation Phosphorylation catalyzed by protein kinases Dephosphorylation catalyzed by protein phosphatases Immediate but slower and longer-lasting effects  Covalent modification phosphorylation - dephosphorylation adenylation - deadenylation methylation - demethylation uridylation - deuridylation ribosylation - deribosylation acetylation - deacetylation  Genetic control of enzyme activity Enzyme levels can be controlled by controlling expression of gene Inducible genes may be turned on/off Involves induction or repression of enzyme synthesis by DNA binding regulatory proteins Hours to days response Exemplified by Lac operon  Genetic control of enzyme activity Lac operon encodes for enzyme lactase Enzyme lactase is produced only if substrate lactose is present Lac operon is turned off without substrate lactose Genetic control of enzyme activity PROMOTER OPERATOR LAC Z ON switch OFF switch Codes for enzyme LACTASE Lac Operon Genetic control of enzyme activity PROMOTER OPERATOR LAC Z REPRESSOR RNA Pol Genetic control of enzyme activity PROMOTER OPERATOR LAC Z REPRESSOR RNA Pol Genetic control of enzyme activity PROMOTER OPERATOR LAC Z REPRESSOR SILENCED RNA Pol No Lactase Genetic control of enzyme activity PROMOTER OPERATOR LAC Z REPRESSOR RNA Pol lactose Genetic control of enzyme activity PROMOTER OPERATOR LAC Z REPRESSOR RNA Pol Genetic control of enzyme activity PROMOTER OPERATOR LAC Z REPRESSOR RNA Pol Genetic control of enzyme activity PROMOTER OPERATOR LAC Z REPRESSOR RNA Pol Genetic control of enzyme activity PROMOTER OPERATOR LAC Z RNA Pol REPRESSOR Genetic control of enzyme activity PROMOTER OPERATOR LAC Z RNA Pol REPRESSOR EXPRESSED (+) Lactase  Genetic control of enzyme activity Without lactose, repressor binds to operator RNA polymerase is unable to bind to promoter Lac operon NOT transcribed into mRNA and NOT translated in lactase  Genetic control of enzyme activity When (+)lactose, it binds to active repressor protein Repressor is inactivated RNA polymerase is now able to bind to promoter region Lac operon transcribed into mRNA mRNA translated into lactase  Summary Enzymes are biologic catalysts Lower energy of activation Enzyme activity affected by pH, temperature and substrate concentration  Summary Regulation includes Storage as zymogen Allosteric regulation Covalent modification Enzyme induction through gene regulation  End of Part 1 Lecture will resume in Time is up Return to your seats  Part 2 Enzyme Kinetics and Inhibition  Lecture outline Part 1: General Part 2: Enzyme Concepts Kinetics and Inhibition Enzyme characteristics Concept of rates, and mechanism of action reaction rates and kinetics Enzyme structure Michaelis-Menten Curve Enzyme nomenclature and classification Lineweaver-Burk Graph Enzyme regulation Enzyme Inhibition  At the end of your study, you are expected to.... Discuss and explain reaction orders Discuss and interpret Michaelis-Menten curve, Lineweaverburk graph Determine Km and Vmax and interpret their significance and meaning Explain types of inhibition Determine and differentiate inhibition type based on Km and Vmax What is RATE? What is rate? It is a measure of change over a period of time What is rate? It is a measure of change over a period of time r s e te m 10 10 meters in 5 seconds Rate = 2m/sec  What is REACTION RATE?  What is reaction rate? Rate: change/time Reaction rate: formation of product over a period of time TIME reactant product d[P] ———- dT 1mL  What is reaction rate? Rate: change/time Reaction rate: formation of product over a period of time TIME reactant product d[P] ———- dT 1mL  What is reaction rate? Rate: change/time Reaction rate: formation of product over a period of time TIME reactant product d[P] ———- dT 1mL  What is reaction rate? Rate: change/time Reaction rate: formation of product over a period of time TIME reactant product d[P] ———- dT 1mL  What is reaction rate? Rate: change/time Reaction rate: formation of product over a period of time TIME reactant product d[P] ———- dT 1mL  What is reaction rate? Rate: change/time Reaction rate: formation of product over a period of time TIME reactant product d[P] ———- dT 1mL  What is reaction rate? Rate: change/time Reaction rate: formation of product over a period of time TIME reactant product d[P] ———- dT 1mL  What is reaction rate? Rate: change/time Reaction rate: formation of product over a period of time TIME reactant product d[P] ———- dT 1mL  What is reaction rate? Rate: change/time Reaction rate: formation of product over a period of time TIME reactant product d[P] ———- dT 1mL  What is reaction rate? Rate: change/time Reaction rate: formation of product over a period of time TIME reactant product d[P] ———- dT 1mL  What is reaction rate? Rate: change/time Reaction rate: formation of product over a period of time TIME reactant product What is concentration of products after 10 seconds? 1mL 10 particles/ml/10sec Reaction rate is 1part/ml/sec  Zero Order Versus First Order 100 100 75 75 Vo 50 50 25 25 zero order first order 0 0 0 1 10 100 1000 0 1 10 100 1000 [R] [R]  Michaelis-Menten Curve V0 mmol/L/sec 20 15 Zero order 10 Mixed order 5 First order 10 20 30 40 60 [S] mmol/L  [S] V0 mmol/L mmol/L/sec S 1  [S] V0 mmol/L mmol/L/sec P 1 1  [S] V0 mmol/L mmol/L/sec S 1 1 2 S  [S] V0 mmol/L mmol/L/sec P 1 1 2 2 P  [S] V0 mmol/L mmol/L/sec S 1 1 2 2 S 5 S S S  [S] V0 mmol/L mmol/L/sec P 1 1 2 2 P 5 5 P P P  [S] V0 mmol/L mmol/L/sec S S 1 1 2 2 S S S 5 5 S 10 S S S S  [S] V0 mmol/L mmol/L/sec P P 1 1 2 2 P P P 5 5 P 10 10 P P P P  [S] V0 mmol/L mmol/L/sec S S S S 1 1 2 2 S S S S 5 5 S S S S 10 10 20 S S S S S S S S  [S] V0 mmol/L mmol/L/sec P P P P 1 1 2 2 P P P P 5 5 P P P P 10 10 20 20 P P P P P P P P  [S] V0 mmol/L mmol/L/sec S S S S S S S S 1 1 2 2 S S S S S S S S 5 5 S S S S S S 10 10 S S 20 20 S S S S S S S 40 S S S S S S S S S  [S] V0 mmol/L mmol/L/sec S S S P P P P S 1 1 2 2 S S P P P P S S 5 5 S P P P P S 10 10 S S 20 20 S S P P P P S 40 20 S S P P P P S S S  [S] V0 S S S mmol/L mmol/L/sec S S S S S S S 1 1 S S S S S S S 2 2 S S S S S 5 5 S S S S S S S S S S 10 10 S S S S S S S 20 20 S S S S S S S S 40 20 S S S S S S S S S 60 S S S S S  [S] V0 S S S mmol/L mmol/L/sec S S S P P P P 1 1 S S S S S S P 2 2 P P P S S 5 5 S S S S P S P P P S 10 10 S S S S S S S 20 20 P P P P S S S S 40 20 S S S S P S P P P 60 20 S S S S S  [S] V0 mmol/L mmol/L/sec 1 1 Michaelis-Menten Curve 2 2 20 5 5 V0 15 mmol/L/sec 10 10 10 5 20 20 10 20 30 40 60 40 20 [S] mmol/L 60 20  [S] V0 mmol/L mmol/L/sec 1 1 Michaelis-Menten Curve 2 2 20 5 5 V0 15 mmol/L/sec 10 10 10 5 20 20 10 20 30 40 60 40 20 [S] mmol/L 60 20  Michaelis-Menten Curve V0 mmol/L/sec 20 15 10 5 10 20 30 40 60 [S] mmol/L  Michaelis-Menten Curve V0 mmol/L/sec 20 15 10 First Order Kinetics 5 10 20 30 40 60 [S] mmol/L  V0 mmol/L/sec 20 15 10 First Order Kinetics 5 10 20 30 40 60 [S] mmol/L  V0 mmol/L/sec 20 15 10 First Order Kinetics 5 10 20 30 40 60 [S] mmol/L  V0 mmol/L/sec 20 15 10 First Order Kinetics 5 10 20 30 40 60 [S] mmol/L  V0 mmol/L/sec 20 Zero Order Kinetics 15 10 5 10 20 30 40 60 [S] mmol/L  V0 mmol/L/sec 20 Zero Order Kinetics 15 10 5 10 20 30 40 60 [S] mmol/L  V0 mmol/L/sec 20 Zero Order Kinetics 15 10 5 10 20 30 40 60 [S] mmol/L  V0 mmol/L/sec 20 Zero Order Kinetics 15 10 5 10 20 30 40 60 [S] mmol/L Michaelis-Menten Curve At low [S], obeys 1st order kinetics More enzymes than substrate Addition of more substrate increases reaction rate V0 All substrates are being converted to products Michaelis-Menten Curve At high [S], obeys zero order kinetics All active sites are occupied Enzyme is saturated Maximum reaction velocity (Vmax) Further addition of substrate CAN NOT increase rate beyond Vmax. Michaelis-Menten Curve Vmax Michaelis-Menten Curve Vmax Vmax1/2 Michaelis-Menten Curve Vmax Vmax1/2 Km Michaelis-Menten Curve Km is Michaelis constant [S] at Vmax1/2 Measure of how well a substrate complexes enzyme Binding affinity Low Km means high binding affinity and vice versa  Low Km vs High Km Vmax V0 mmol/L/sec Vmax1/2 Km1 Km2 [S] mmol/L 1 Km=5 2 Km=10 3 Km=15  Michaelis-Menten Curve V0 mmol/L/sec Vmax1/2 [S] mmol/L  Michaelis-Menten Lineweaver-Burke equation equation [S] vs. V0 1/[S] vs. 1/V0 Nonlinear, hyperbolic Linear Difficult to estimate Km and Vmax easily Km extrapolated  How to convert Micahelis-Menten equation to Lineweaver-Burk formula  How to convert Micahelis-Menten equation to Lineweaver-Burk formula 1. Obtain reciprocal 2. Separate Km and [S] 3. Cancel and simplify y = mx + b Lineweaver-Burk Plot y = mx + b  Determine Vmax and Km X Y PROBLEM You want to determine the Vmax and Km [S] V0 for a particular enzyme. You decided to measure the reaction rate V0 at different substrate concentrations [S]. This 10 33 generated the date shown on right which you decided to use in constructing 5 25 Michaelis-Menten Curve. 3.3 20  Determine Vmax and Km X Y 40 [S] V0 30 10 33 V0 20 5 25 10 3.3 20 2 4 6 8 10 [S] Are we sure Vmax is around Michaelis-Menten Curve 33-35? Determine Vmax and Km X Y X Y Obtain [S] V0 1/[S] 1/V0 reciprocal 10 33 0.1 0.03 5 25 0.2 0.04 3.3 20 0.3 0.05  Determine Vmax and Km X Y 0.05 0.04 1/[S] 1/V0 1/V0 0.03 0.1 0.03 0.02 0.01 0.2 0.04 -0.2 -0.1 0.1 0.2 0.3 0.4 0.3 0.05 1/[S] Lineweaver-Burk Plot  Determine Vmax and Km X Y 0.05 0.04 1/[S] 1/V0 1/V0 0.03 0.1 0.03 0.02 0.01 0.2 0.04 -0.2 -0.1 0.1 0.2 0.3 0.4 0.3 0.05 1/[S] Lineweaver-Burk Plot  Determine Vmax and Km X Y 0.05 0.04 1/[S] 1/V0 1/V0 0.03 0.1 0.03 0.02 1/Vmax 0.01 0.2 0.04 -0.2 -0.1 0.1 0.2 0.3 0.4 0.3 0.05 1/[S] Lineweaver-Burk Plot  Determine Vmax and Km X Y 0.05 0.04 1/[S] 1/V0 1/V0 0.03 0.1 0.03 0.02 0.01 0.2 0.04 -0.2 -0.1 0.1 0.2 0.3 0.4 0.3 0.05 1/[S] Lineweaver-Burk Plot  Determine Vmax and Km X Y 0.05 0.04 1/[S] 1/V0 1/V0 0.03 -1/Km 0.1 0.03 0.02 0.01 0.2 0.04 -0.2 -0.1 0.1 0.2 0.3 0.4 0.3 0.05 1/[S] Lineweaver-Burk Plot  Recap Michaelis-Menten Lineweaver-Burk [S] vs V0 1/[S] vs 1/V0 Hyperbolic curve Linear  Enzyme inhibition Inhibition Covalent binding Noncovalent binding Irreversible Reversible Competitive Noncompetitive Competitive Noncompetitive Vmax unchanged Vmax decreased Viagra Penicillin Km increased Km unchanged Cyanide  Reversible competitive inhibition Substrate and Inhibitor are competing for ACTIVE SITE S E I  Reversible competitive inhibition Substrate and Inhibitor are competing for ACTIVE SITE E S I When substrate bind first, ES complex and products can be formed  Reversible competitive inhibition Substrate and Inhibitor are competing for ACTIVE SITE S E I  Reversible competitive inhibition Substrate and Inhibitor are competing for ACTIVE SITE E I S However when Inhibitor binds first, NO ES complex nor products can be formed  Reversible competitive inhibition Substrate and inhibitor compete for active site Inhibitor blocks active site Competitive inhibitors often are structural analogues of substrate (BUT NOT ALWAYS) How is competitive inhibition overcome? Reversible competitive inhibition S E I Reversible competitive inhibition S E I Reversible competitive inhibition S S E S S I S  Reversible competitive inhibition At higher [S], ES complex and products can be formed S S S E S I S  Reversible competitive inhibition Vmax V0 Vmax1/2 Km Km NO INHIBITOR W/ INHIBITOR [S]  Reversible competitive inhibition Vmax may be attained at higher [S] Vmax is unchanged Km is apparently increased  Reversible competitive inhibition S S ENZYME ENZYME ENZYME ENZYME  Reversible competitive inhibition S S ENZYME ENZYME ENZYME ENZYME Km = 2  Reversible competitive inhibition S S I I I I ENZYME ENZYME ENZYME ENZYME  Reversible competitive inhibition S I I I ENZYME ENZYME S I ENZYME ENZYME In the presence of Competitive Inhibitor, Km should be > 2  Reversible competitive inhibition S S S I I S I S I ENZYME ENZYME ENZYME ENZYME In the presence of Competitive Inhibitor, Km should be > 2  Reversible competitive inhibition I S I S S S S ENZYME ENZYME I I ENZYME ENZYME In the presence of Competitive Inhibitor, Km should be > 2 Reversible noncompetitive inhibition Substrate and Inhibitor have different binding sites S Active site E Allosteric site I  Reversible noncompetitive inhibition Substrate and Inhibitor have different binding sites E S I EIS complex is inactive and products can NOT be formed  Reversible noncompetitive inhibition Inhibitor can bind to the enzyme at the same time as the substrate Inhibitors bind to allosteric site Both the EIS and EI complexes are enzymatically inactive Can increasing [S] overcome inhibitor? Reversible noncompetitive inhibition Substrate and Inhibitor have different binding sites S S S Active site E S Allosteric site S I S  Reversible noncompetitive inhibition Substrate and Inhibitor have different binding sites S S E S S I S S EIS complex is inactive and products can NOT be formed Reversible noncompetitive inhibition Vmax (No Inhibitor) V0 Vmax (W/ Inhibitor) Km [S]  Reversible noncompetitive inhibition Vmax can not be attained even at higher [S] Vmax is decreased Km is unchanged  Reversible noncompetitive inhibition S S ENZYME ENZYME ENZYME ENZYME  Reversible noncompetitive inhibition S S ENZYME ENZYME ENZYME ENZYME Km = 2  Reversible noncompetitive inhibition S S ENZYME ENZYME INHIBITOR INHIBITOR ENZYME ENZYME INHIBITOR INHIBITOR  Reversible noncompetitive inhibition S S ENZYME ENZYME INHIBITOR INHIBITOR ENZYME ENZYME INHIBITOR INHIBITOR In the presence of Noncompetitive Inhibitor, Km is unchanged Competitive versus noncompetitive in Lineweaver-Burk plot 1/V0 (+)Inhibitor 1/V0 (+)Inhibitor No Inhibitor No Inhibitor 1/[S] 1/[S] Competitive Noncompetitive  Enzyme inhibition summary tabulation Inhibition type Binding site Vmax Km Competitive Active site No change Increase Non-competitive Allosteric site Decrease No change  Drugs that are enzyme inhibitors Many drugs work by inhibiting enzymes Can slow disease progression or manage symptoms Examples: Statins (lower cholesterol) ACE inhibitors (treat high blood pressure) Aspirin (analgesic)  Enzyme deficiency may cause disease/disorders Not enough enzyme to function correctly. Consequence: Buildup of substances the enzyme normally breaks down. Outcome: Various diseases and disorders. Examples: Phenylketonuria (PKU) & Glucose-6- phosphate dehydrogenase (G6PD) deficiency. Phenylketonuria  Phenylketonuria Genetic disorder, autosomal recessive Deficiency of phenylalanine hydroxylase Accumulation of Phe and its metabolites in tissues and body fluids Mental retardation Prevalence of PKU is approximately 1 in 10,000 in European populations  Mousy odor Mental retardation  G6PD deficiency Genetic disorder, X-linked recessive Deficiency of glucose-6-phosphate dehydrogenase Faiulure to neutralize free-radicals Hemolysis Affects about 400 million people worldwide  Keypoints to remember Biological catalysts Made of protein Speed up chemical reactions by decreasing energy of activation Not consumed in the reaction  Keypoints to remember Lock-and-key model: Enzyme has an active site that fits a specific substrate Induced fit model: Enzyme slightly changes shape upon substrate binding  Keypoints to remember Six main classes: Oxidoreductases: Transfer electrons Transferases: Transfer functional groups Hydrolases: Break down molecules using water Lyases: Cleave bonds by means other than hydrolysis Isomerases: Rearrange atoms within a molecule Ligases: Form new bonds between two molecules  Keypoints to remember Temperature: Enzymes have an optimal temperature range for activity pH: Enzymes have a specific pH range for optimal function Inhibitors: Molecules that decrease enzyme activity  Keypoints to remember Drugs as Enzyme Inhibitors: Many medications work by inhibiting specific enzymes. By blocking the enzyme's activity, they can alter a disease process.  Keypoints to remember Genetic mutations can lead to enzyme deficiencies. When a crucial enzyme is deficient, the corresponding biochemical reaction slows down or stops. This can lead to various diseases.  Which of these statements about enzyme-catalyzed reactions that obey Michaelis-Menten kinetics is TRUE? A. At enzyme saturation, the reaction rate is independent of substrate concentration. B. The Michaelis-Menten constant Km equals the [S] at which V0 = Vmax. C. If enough substrate is added, the normal Vmax of a reaction can be attained even in the presence of a noncompetitive inhibitor. D. The rate of a reaction increases steadily with time as substrate is depleted. E. The activation energy for the catalyzed reaction is the same as for the uncatalyzed reaction A 55 year-old male presents with severe xanthomas. Laboratory examination reveals elevation of serum cholesterol. A statin drug that inhibits the rate limiting enzyme of cholesterol biosynthesis was given to control the hypercholesterolemia. By inhibiting this enzyme, which of the following will change about the reaction it catalyzes? A. The energy of activation will be higher in the presence of the drug B. There will be a higher net free energy change in the presence of the drug C. There will be a lower net free energy change in the presence of the drug D. Lower equilibrium concentration of the HMG-CoA in the presence of the drug E. Higher concentration of mevalonate in the presence of the drug  A 55 year-old male presents with difficulty breathing and swollen ankles. He is found to have a failing heart, resulting in blood backing up to his lungs and making it difficult for him to breathe. He is administered a drug that inhibits Angiotensin Converting Enzyme. The drug cited above acts by lowering the Vmax without affecting the binding affinity between Angiotensin Converting Enzyme and its substrate. What is the mechanism of action of the drug? A. Competitive inhibition of enzyme; drug binds to active site B. Non-competitive inhibition of enzyme: drug binds to allosteric site C. Non-competitive inhibition of enzyme; drug binds to active site D. Competitive inhibition of enzyme: drug binds to allosteric site E. Uncompetitive inhibition of ES complex; drug binds to allosteric site of ES complex  Below is a list of five substrates and their corresponding Km values for enzyme X. Based on this information which of the substrates binds tightest to the enzyme? A. substrate A (Km = 2.1 X 10-6) B. substrate B (Km = 5.4 X 10-4) C. substrate C (Km = 7.0 X 10-6) D. substrate D (Km = 1.5 X 10-5) E. substrate E (Km = 1.5 X 10-9) B I O C H E M E N Z Y M E I S N O T H A R D Y E S. I C A N. T EHD NE  E A SY T HE. I N END. C  References Lippincott’s Illustrated Reviews Biochemistry 26th edition Harper’s Illustrated Biochemistry 28th edition Lehninger’s Principle of Biochemistry 4th ed  Enzyme classification Class Type of reaction Oxidoreductase Transfer of electrons Transferase Group transfer reactions Hydrolase Hydrolytic reactions Lyase Addition/ removal of groups Isomerase Structural rearrangement Ligase Condensation reaction coupled to ATP hydrolysis  How are enzymes classified? 1. Oxidoreductases... [ dehydrogenases] catalyzes oxidation reduction reactions, often using coenzyme as NAD+/FAD TIP: Usually found in energy releasing pathways Alcohol dehydrogenase e- Lehninger’s Principle of Biochemistry 4th ed page 199  How are enzymes classified? 2. Transferases... catalyze the transfer of functional group TIP: Usually key control enzymes are kinases Hexokinase Lehninger’s Principle of Biochemistry 4th ed page 199  How are enzymes classified? 3. Hydrolyases… catalyzes hydrolytic reactions adds water across C-C bonds TIP: Proteolytic and other digestive enzymes Lehninger’s Principle of Biochemistry 4th ed page 199  How are enzymes classified? 4. Lyases... cleave C-C, C-O, C-N & other bonds often generating a C=C bond or ring Pyruvate decarboxylase Lehninger’s Principle of Biochemistry 4th ed page 199  How are enzymes classified? 5. Isomerases… [mutases] catalyze isomerizations Maleate isomerase Lehninger’s Principle of Biochemistry 4th ed page 199  How are enzymes classified? 6. Ligases… condensation of 2 substrates with splitting of ATP Pyruvate Carboxylase Lehninger’s Principle of Biochemistry 4th ed page 199  Michaelis constant Vmax 5.0 x 10-50 1.5 x 10-20 Reverse transcriptase mmol/uL mmol/uL/sec Reverse transcriptase 5.0 x 10-5 1.5 x 10-20 plus Drug mmol/uL mmol/uL/sec  Michaelis constant Vmax 5.0 x 10-5 1.5 x 10-20 Enzyme mmol/uL mmol/uL/sec 5.0 x 10-5 1.5 x 10-70 Enzyme plus Drug mmol/uL mmol/uL/sec  Vmax Michaelis constant 5.0 x 10-5 1.5 x 10-20 Enzyme mmol/uL/sec mmol/uL 5.0 x 10-5 1.5 x 10-5 Enzyme plus Inhibitor mmol/uL/sec mmol/uL  Vmax Michaelis constant 5.0 x 10-5 1.5 x 10-20 Enzyme mmol/uL/sec mmol/uL 5.0 x 10-15 1.5 x 10-5 Enzyme plus Inhibitor mmol/uL/sec mmol/uL  Vmax Michaelis constant 5.0 x 10-15 1.5 x 10-2 Enzyme mmol/uL/sec mmol/uL 5.0 x 10-5 1.5 x 10-20 Enzyme plus Inhibitor mmol/uL/sec mmol/uL  0.05 0.04 1/V0 0.03 0.02 0.01 0.1 0.2 0.3 0.4 1/[S]  0.05 0.04 1/V0 0.03 0.02 0.01 -0.2 -0.1 0.1 0.2 0.3 0.4 1/[S]  0.05 0.04 1/V0 0.03 0.02 0.01 -0.2 -0.1 0.1 0.2 0.3 0.4 1/[S]  0.05 0.04 1/V0 0.03 0.02 0.01 -0.2 -0.1 0.1 0.2 0.3 0.4 1/[S]  0.05 0.04 1/V0 0.03 0.02 0.01 -0.2 -0.1 0.1 0.2 0.3 0.4 1/[S]  0.05 0.04 1/V0 0.03 0.02 0.01 -0.2 -0.1 0.1 0.2 0.3 0.4 1/[S]  0.05 0.04 1/V0 0.03 0.02 0.01 -0.2 -0.1 0.1 0.2 0.3 0.4 1/[S] 1 = V0 + 1 Vmax = Km [S] Vmax Isoenzyme 1 Isoenzyme 2 Isoenzyme 3  0.05 0.04 1/V0 0.03 0.02 0.01 0.1 0.2 0.3 0.4 1/[S]