Basics of Enzymes PDF

Basics of Enzymes and Enzyme Regulation Farzaneh Daghigh, Ph.D. Evans 504 Virtual Office Hours: M- Th @ 4-5 PM See the instructions in the note below to schedule a virtual appointment ...

Basics of Enzymes and Enzyme Regulation Farzaneh Daghigh, Ph.D. Evans 504 Virtual Office Hours: M- Th @ 4-5 PM See the instructions in the note below to schedule a virtual appointment  Learning Objectives 1. Explain the process of substrate molecules being enzymatically transformed into products (including transition state complex and activation energy) and illustrate the process on an energy diagram 2. Describe lock and key and induced fit 3. Differentiate between cofactors, coenzymes, and isozymes 4. Identify the different functional groups (amino acid side chains, coenzymes, and metal ions) and explain how each contributes to an enzymatic reaction 5. Describe factors (pH, temperature, concentrations of enzyme and substrate) affecting the rate of a reaction 6. Compare and contrast between Michaelis-Menten and Lineweaver Burk plots for enzyme inhibition, describe where K M, Vmax, KM,app, Vi are present on each plot, and predict the effect of reaction perturbations (i.e., increases in substrate, increases in inhibitor, etc.) on each plot 7. Contrast KD with KM 8. Explain the importance of a regulatory enzyme in a metabolic pathway using examples 9. Compare and contrast the mechanisms for regulating enzymes (feedback inhibition, allosteric, hormonal regulation via covalent modification, and induction/repression), and relate to the time required for each 10. Compare the catalytic mechanism of an allosteric enzyme to an enzyme that follows Michaelis-Menten kinetics  What are Enzymes? Enzymes: Protein catalysts that increase the rate of the reactions without being changed in the overall process All enzymes are proteins Exception: Ribozymes Catalytic ribonucleic invoking catalysis in RNA processing  Enzyme Substrate Complexes and Transition States Enzyme-Substrate Complex: combination of enzyme & substrate Rate of an enzyme catalyzed reaction is proportional to the amount of E.S. since product formation occurs after formation of complex Transition State: Highly unstable intermediate which is the E: Enzyme S: Substrate “half-way” point between substrate and product which the ES: Enzyme-Substrate Complex enzyme actively stabilizes to facilitate the T: Transition State reaction. P: Product Enzymes stabilize transition states  Catalytic Efficiency of Enzymes Without an enzyme or a catalyst, a reaction will proceed extremely slowly, if at all The rate of un-catalyzed reactions is slow due to the high free energy of activation Enzymes lower the free energy of activation Enzymes DO NOT change: Enzyme complementary to transition state Free energy of the reactants Free energy of products Reaction equilibrium Lehninger, 3rd ed.  Free Energy of Activation Free Energy of Activation: The difference in free energy between the transition state and the reactant Enzymes change activation energy but NOT the overall energy Enzymes increase the rate of exergonic reactions by lowering the activation energy 2022 Types of Enzyme & Substrate Interactions “Lock and Key” Model “Induced Fit” Model Factors that Enzymes Need for Catalysis 1. Active Sites 2. Cofactors Metal ions 3. Coenzymes Prosthetic groups & Co-substrates 4. Compartmentalization 5. Specificity  Reaction in the Enzyme Active Site Active Site: special pocket containing amino acid side chains that create a 3D surface complementary to the substrate Amino acids lining  the substrate forming weak bonds   Marks’ 3rd ed  Cofactors Compounds that facilitate enzyme reactions Cofactors: Non-protein 1) INORGANIC cofactors: compounds & metal ions i.e. Mg, K, Ca, Zn, Fe, Cu contribute to catalytic process by donating/accepting electrons Bound within enzyme molecule 2) ORGANIC cofactors or Coenzymes Bind temporarily or permanently to enzyme near active site Many vitamins NAD (niacin, B3) FAD (riboflavin, B2) Coenzyme A Two Types: Prosthetic Group: tightly covalently bound coenzyme – doesn’t dissociate from enzyme Co-substrate: associates with enzyme temporarily Water Soluble Vitamin Coenzymes:  Compartmentalization Enzymes are localized in specific organelles within the cells This compartmentalization serves to isolate the reaction substrate or products from other competing reactions  Substrate Specificity Specificity: refers to the ability of an enzyme to discriminate between two competing substrates A highly simplified representation of part of the substrate binding sites in chymotrypsin, trypsin, and elastase (From: Stryer, Biochemistry, fig9-47. , p.226 ) Enzymes are highly specific both in the reaction catalyzed and their choice of reactants (the substrates) An enzyme usually catalyzes a single reaction Side reactions leading to the wasteful formation of by-products rarely occur in enzyme-catalyzed reactions, in contrast with un-catalyzed ones Chymotrypsin cuts the peptide bond on the carboxyl side of aromatic amino acids (phenylalanine, tyrosine, or tryptophan) Trypsin cuts the peptide bond on the carboxyl side of basic amino acids (arginine or lysine) Elastase cuts the peptide bond on the carboxyl side of smaller, uncharged amino acids (glycine, alanine, serine)  Types of Enzymes Isoenzyme Isoenzyme: multiple forms of an enzyme catalyzing the same reaction  Types of Enzymes Allosteric Enzymes Allosteric Enzyme: Multi-subunit enzymes with identical or different polypeptide chains https://ib.bioninja.com.au/higher-level/topic-8-metabolism-cel  Types of Enzymes Proenzymes Proenzymes: The inactive precursor form of an enzyme. Cleavage of a specific peptide bond within the proenzyme generates the active mature enzyme.  Variables the Affect the Activity of an Enzyme Reaction pH Temperature Substrate Concentration/ Enzyme Concentration  Enzyme Activity pH pH: measure of the acidity or alkalinity of a solution Enzymes have evolved to operate at specific pH values and deviation from this value will lead to a decrease in enzyme activity A too high or low pH can denature enzymes just as high temperatures do Most of our bodily fluids have a neutral pH of approximately 7.2, therefore human enzymes have the highest activity at this pH [Life: The Science of Biology, Purves, 4th Edition]  Enzyme Activity Temperature As temperature increases, enzyme activity also increases because there is an increase in the number of collisions between the reacting molecules and the enzymes. Increasing the temperature further leads to a peak in enzyme activity. For human enzymes, this peak temperature is approximately 98.6 degrees Fahrenheit, which is our body temperature. Any further increase in temperature leads to a decrease in the enzyme activity due to the denaturing of the enzyme protein Rates double for every 10 degree increase in temperature Credit: Ben Himme at Pathwayze.com  Enzyme Kinetics Kinetics measures how fast the reaction takes place (its velocity or speed) Shout out to: The rate of the reaction is influenced by pH, temperature of the reaction, the substrate concentration, and the enzyme concentration [Tuition Tube, The Michaelis-Menten Equation in Biochemistry]  Enzyme Kinetics Keeping pH, temperature and concentration of enzyme constant: Increasing the substrate concentration increases the initial rate (velocity, or activity) of the reaction Enzyme activity can be depicted in a plot of [Marks’ Basic Med. Biochemistry, 5th Ed., Fig. 9.2] substrate concentration vs. initial rate of reaction A plot of the reaction velocity, V, as a function of the substrate concentration, [S], for an enzyme that obeys Michaelis-Menten kinetics  Enzyme Kinetics: KM KM is numerically equal to the substrate concentration at which the reaction velocity is equal to ½ Vmax KM does not vary with enzyme concentration Saturation kinetics refers an enzyme reaction reaching a maximal velocity at high levels of S Representation of an enzyme substrate complex at how (A), at high (C), and at Km concentration of substrate (B) Important Information from Michaelis – Menten Kinetics Comparison of Two Isoenzymes: Hexokinase and Glucokinase Catalyze the transfer of a phosphate group from ATP to glucose Meisenberg & Simmons. Principles of Medical [Lippincott Biochemistry, 5th Ed., Fig. 5.9]  Hexokinase Found in all cells but very little in liver cells Has broad specificity (glucose or fructose) The KM of hexokinase for glucose is 0.1 mM – high affinity for glucose Ensures a supply of glucose for tissues even when the blood glucose concentration is low  Glucokinase Found mainly in the liver parenchymal cells and pancreatic islet cells Glucokinase only acts on glucose KM of glucokinase is 10mM Function is to remove glucose from the blood following a meal  KM versus KD KM - Michaelis-Menten constant KD - Dissociation constant Kinetic constant Thermodynamic constant Measures the impact of substrate True measure of the affinity of concentration on the speed of a a ligand for a binding site of an reaction enzyme Can be used as an indirect i.e.: concentration 50% of measure of affinity in the active ligand will dissociate from site the enzyme Note: doesn’t address speed of the reaction allosteric site Note: for both KM and KD, the smaller allosteric site the number, the bigger the affinity!!  Compare and Contrast KD and KM KD vs KM KD is the dissociation constant. KM is the Michaelis constant. Nature KD is a thermodynamic constant. An KM is a kinetic constant. Not an equilibrium constant equilibrium constant Details KM represents the affinity of KD represents the affinity of a ligand substrates binding for the active site. It shows the relationship (such as an allosteric effector) towards between substrate concentration an enzyme. It reports the true affinity of and reaction speed and how quickly a ligand for binding a site on protein. the enzyme-substrate complex is turned over into product Similarities: for both KM and KD, the smaller the number, the bigger the affinity!! Enzyme Regulation Enzymes play a crucial role in cellular metabolism and are tightly regulated to adapt to changing cellular demands and conditions. Regulation by other molecules that either increase or reduce their activity: Molecules that increase the activity of an enzyme are called activators, while molecules that decrease the activity of an enzyme are called inhibitors There are many kinds of molecules that block or promote enzyme function, & that affect enzyme function by different routes Enzyme Regulation What alters an enzyme’s rate? Substrates / Inhibitors Allosteric regulation of enzymes Covalent modification of enzymes via hormonal regulation  Enzyme Regulation Substrates/Inhibitors Substrate Availability: The velocity will change, depending on how much substrate is present, and the effect is immediate (< sec) Product Inhibition (Feedback Loops): VMAX and/or Km will change, depending on whether the inhibitor is competitive or noncompetitive. Since inhibitors may be present in the cell, or in the blood stream, the effect is immediate (< sec) [Cornell, B. 2016. Feedback Inhibition]  Enzyme Regulation Allosteric Enzymes Allosteric enzymes Oligomers Multiple active sites Change their conformation upon binding of an allosteric effector Show cooperative binding Display a sigmoidal dependence on the concentration of their substrates Allows them to greatly vary catalytic output in response to small changes in effector or ligand concentration Follow Sigmoidal plot instead of hyperbolic when plotted as velocity vs. substrate concentration Allosteric enzymes do not obey Michaelis – Menten kinetics  Enzyme Regulation Allosteric Enzymes Allosteric Effectors: Activators or Inhibitors Small molecules that bind to the enzyme at sites other than the active site and regulate enzymatic activity. Activators tend to bind more tightly to R state Inhibitors tend to bind more tightly to T state [OpenStax Biology, Chapter 6.5: Enzymes, Fig. 4]  Enzyme Regulation Allosteric Enzymes One of the best examples of allosteric effects is hemoglobin Hemoglobin Each of the 4 subunits binds one heme molecule Myoglobin Resembles one subunit of hemoglobin  Enzyme Regulation Allosteric Enzymes [Cornell, B. 2016. Cooperative Binding of Hemoglobin] The sigmoidal shape of the curve is due to a conformational change in the enzyme Enzyme Regulation Covalent Modification Covalent Modification: Another enzyme can add a phosphate or otherwise covalently alter the enzyme, causing the enzyme to be more or less active These effects can be immediate or can take minutes to occur. [Lippincott Biochemistry, 5th Ed., Fig. 5.9]  Enzyme Regulation Summary Regulator Event Typical Effectors Results Time for Change Substrate availability Substrate Change in velocity Seconds Product inhibition Product Change in Vm &/or Km Seconds Allosteric control End product Change in Vm &/or Km Seconds Covalent modification Another enzyme Change in Vm &/or Km Seconds to minutes Induction or Repression Hormone/ metabolite Change in the amount Minutes to hours to days What Alters an Enzyme’s Levels or Concentration Induction or Repression [Molec. Biology of the Cell, Alberts, 5th Ed., Fig. 15-6] Name the Type of Regulation Inhibition Reversible vs. Irreversible Competitive Inhibitors Resemble the substrate and bind reversibly at the active site Form an enzyme-inhibitor complex Noncompetitive Inhibitors Bind to the enzyme reversibly in a different domain than the active site Change the conformation of the enzyme Inhibition Competitive Inhibitor Competitive inhibitor raises the apparent Km for the substrate, without changing the maximum rate of reaction [Med. Biochemistry: An Illustrated Review, Thieme, 1st Ed., Fig. 5.7A] Inhibition Non-competitive Inhibitor Noncompetitive inhibitor lowers the Vmax, but does NOT affect the Km for the substrate [Med. Biochemistry: An Illustrated Review, Thieme, 1st Ed., Fig. 5.7B] Inhibition Uncompetitive Inhibitor Inhibitor binds to the ES Complex & cannot be overcome by addition of a substrate Vmax and Km decrease i.e. Alkaline Phosphatase by Phenylalanine Slidesharecdn.com Summary of the Types of Inhibition  [Lippincott Biochemistry, 5th Ed., Fig. 5.13] Competitive Inhibition Example  Example Non-Competitive Inhibition  Example Non-Competitive Inhibition Silver Poisoning [Clark, 2007] [Biochemistry for Medics, Chhabra, 2015] Example Uncompetitive Inhibition 1. The inhibition of aryl sulphatase by hydrazine 2. The inhibition of intestinal alkaline phosphatase by phenylalanine Irreversible (Suicide) Inhibitors An irreversible inhibitor binds very tightly to the active site of the enzyme often forming covalent bonds with the enzyme to permanently inactivate the enzyme. End result : Fewer enzyme molecules active to catalyze normal reactions– the Vmax decreases Suicide inhibitors are useful as new pharmaceutical agents. [Med. Biochemistry: An Illustrated Review, Thieme, 1st Ed., Fig. 5.7C] Example of Suicide Inhibitor [Enzkinetics, 2014] Example of Suicide Inhibitor Penicillin inhibits bacterial peptidoglycan synthesis Regulatory Enzymes Catalyze irreversible metabolic reactions Catalyze the first committed step in a metabolic pathway Regulation of each step permits efficient regulation of flux of metabolites through the pathway  Regulatory Enzymes Phosphofructokinase Allosteric regulatory enzyme: sigmoidal response to its substrate, fructose-6-phosphate Activated by fructose-2,6-P Inhibited by citrate The ”Rate Limiting Step” of glycolysis Rate Limiting Enzyme [Marks’ Basic Med. Biochemistry, 5th Ed., Fig. 9.1] Nutrition and Enzymes Endogenous: Digestive enzymes (Protease, Lipase, & amylase) Autolytic enzymes Enzyme production (younger vs. older) Exogenous: animal & plants High temp destroys enzymes Cooked vs. raw food Good sources papaya, pineapple, and sprouts Study Questions 1. A 16-year old girl visits her pediatrician with complaints of muscle weakness in her legs and drooping of her left eyelid after moderate physical activity. Nerve stimulation tests reveal a defect in neurotransmitter signaling at the neuromuscular junctions in her legs. Her doctor prescribes physostigmine which is a cholinesterase inhibitor, inhibiting acetylcholinesterase, which normally breaks down acetylcholine in the synaptic cleft. How does this drug alleviate her symptoms? A. increases the acetylcholine concentration in the neuromuscular junctions B. increases the affinity of acetylcholine esterase for acetylcholine C. increases the Vmax of acetylcholine esterase D. increases the Km of acetylcholine esterase for its receptor 2. Aspirin covalently acetylates a serine residue at the active site of cyclooxygenase (COX) isozymes. What best describes effects of aspirin on COX enzymes? A.competitive inhibitor B.reversible inhibitor C.irreversible inhibitor D.transition-state analog 3. What best explains the mechanism of action of methotrexate (a folate analog) on dihydrofolate reductase (DHFR) enzyme in the synthesis of tetrahydrofolate (TH4)? A. Methotrexate decreases the affinity of DHFR for folate B. Methotrexate increases the affinity of DHFR for folate C. The maximum reaction rate for TH4 formation is decreased by Methotrexate D. The maximum reaction rate for TH4 formation is increased by Methotrexate One mechanism virtually all enzymes use to catalyze reactions is: A. Stable binding to the substrate B. Stable binding to the transition state C. Stable binding to the product D. Adding ATP to the reaction to provide energy E. Remove the product to help pull the reaction along