Enzymes PDF

ENZYMES John Paul Martin D. Bagos, MD, FPCP, DPCEDM Assistant Professor, Department of Biochemistry and Nutrition FEU-NRMF Institute of Medicine Learning Objectives Define enzymes. Discuss different enzyme classification. Discuss enzyme structure and function. Discuss e...

ENZYMES John Paul Martin D. Bagos, MD, FPCP, DPCEDM Assistant Professor, Department of Biochemistry and Nutrition FEU-NRMF Institute of Medicine Learning Objectives Define enzymes. Discuss different enzyme classification. Discuss enzyme structure and function. Discuss enzyme kinetics. Discuss factors affecting enzyme activity. Discuss regulation of enzyme activity. Differentiate between different types of reaction orders. Determine inhibition type based on kinetic changes and Vmax. Enzymes: Mechanism of Action Part 1 What are Enzymes? Catalyst of high molecular weight and biological origin. Almost all enzymes are proteins. Exceptions: Ribosomal RNA; RNA with endonuclease (Ribozymes) Accelerate the rate of reactions without undergoing any change themselves. Play a crucial role in numerous regulatory mechanisms, enabling metabolism to adapt to varying conditions. Enzymes Highly efficient Enhance the rates of corresponding noncatalyzed reaction by factors of 10 6 to 109 or even more. Extremely selective Specific for a single substrate or a single stereoisomer (D- vs L- sugars and amino acids) Classification by Reaction Type 1. Oxidoreductases – redox reactions 2. Transferases – transfer of moieties such as glycosyl, methyl, or phosphoryl groups 3. Hydrolases – hydrolytic cleavage of C—C, C—O, C—N, and other covalent bonds 4. Lyases (synthases) – cleavage of covalent bonds by atom elimination, generating double bonds 5. Isomerases – catalyze geometric or structural changes within a molecule 6. Ligases (synthetases) – joining together (ligation) of two molecules in reactions coupled to the hydrolysis of ATP Active Site Site where catalysis occurs. Special pocket or cleft, formed by folding of proteins – participate in substrate binding and catalysis. Substrate are brought in close proximity with cofactors, prosthetic groups, aminoacyl side chains. Nonprotein moieties that participate in catalysis Prosthetic groups Tight and stably incorporated into a protein’s structure by covalent bonds or noncovalent forces Eg. Pyridoxal phosphate, flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), thiamine pyrophosphate, lipoic acid, biotin, transition metals – Fe, Co, Cu, Mg, Mn, Zn Cofactors Similar with prosthetic groups – but bind weakly and transiently to their cognate enzymes or substrates (dissociable complexes) Metal ions Metal activated enzymes vs metalloenzymes (prosthetic group) Nonprotein moieties that participate in catalysis Coenzymes Small organic molecules Derived from water-soluble B vitamins NAD and NADP – nicotinamide FMN and FAD – riboflavin Coenzyme A – pantothenic acid Holoenzyme vs Apoenzyme Holoenzymes is an active enzyme with its nonprotein moiety Apoenzyme is an enzyme without its nonprotein moiety. Mechanism of enzyme action Enzyme bind substrate at active/catalytic site Active site fits shape of substrate Association between enzyme and substrate is temporary E+S ES E+P Four Mechanistic Strategies during catalysis 1. Catalysis by proximity 2. Acid-base catalysis 3. Catalysis by strain 4. Covalent catalysis Catalysis by proximity The higher the concentration, the more frequently substrates will encounter one another and the greater will be the rate at which reaction products appear Acid-base catalysis Ionizable functional groups of aminoacyl side chains contribute to catalysis by acting as acids or bases. Specific acid or base catalysis Participating acids or bases are protons or hydroxide ions Independent of the concentration of other acids or based in the active site General acid or base catalysis Reactions whose rates are responsive to all acids or bases present Eg. HIV Protease Catalysis by strain Creates a conformational change in an enzyme that weaken the bond through physical distortion and electronic polarization Transition state intermediate - strained conformation, midway point in transformation of substrates to products. Covalent catalysis Formation of covalent bond between enzymes and one or more substrates. Modified enzyme becomes a reactant Follows a ping pong mechanism – first substrate is bound and its product is released prior to the bonding of the second substrate. Eg. Chymotrypsin; Fructose-2,6-Bisphosphatase Common in group transfer reactions Conformational changes in enzymes Fischer’s ”lock and key model” Exquisite specificity of enzyme-substrate interactions. Did not account for the for the dynamic changes that accompany catalytic transformation Rigid active site Conformational changes in enzymes Daniel Koshland’s induced fit model Substrates bind to an enzyme inducing conformational changes that is analogous to placing a hand (substrate) into a glove (enzyme).  Please download and install the Slido app on all computers you use Which of the following models of enzyme- substrate binding explains the exquisite specificity of enzyme-substrate interactions? ⓘ Start presenting to display the poll results on this slide. Enzymes: Kinetics Part 2 What is reaction rate? Rate: change/time Reaction rate: formation of product over a period of time Kinetic Theory/Collision Theory - Molecules must approach within bond-forming distance of one another or “collide” - Possess sufficient kinetic energy to overcome energy barrier for reaching the transition state Free energy dictates the direction of chemical reactions Gibbs free-energy change (ΔG) describes the direction in which a chemical reaction will tend to proceed and the concentrations of reactants and products that will be present at equilibrium Products Substrates Enzymes do not affect the equilibrium constant and free energy for the overall reaction Free energy dictates the direction of chemical reactions If ΔG0 is negative number, Keq will be greater than unity, and the concentration of products will exceed that of the substrates (favors product formation). If ΔG0 is positive number, Keq will be less than unity, and the formation of substrates will be favored Activation energy dictates rate of reactions Activation energy is the difference in free energy between the reactant and the transition state. High activation energy (Eact) in uncatalyzed reaction results in slow reaction rate. Lower activation energy, faster rate of reaction More molecules have sufficient energy to pass through the transition state Enzymes provide alternation reaction pathway that lowers activation energy; does not affect free energy of substrates and products. Factors affecting reaction velocity Substrate Concentration Rate of an enzyme-catalyzed reaction increases with substrate concentration until a maximal velocity (V max) is reached. Temperature Increasing temperature increases the rate of both uncatalyzed and enzyme- catalyzed reaction by increasing kinetic energy and the collision frequency of reacting molecules. Higher temperature will result to denaturation resulting loss of the catalytic activity Human enzymes generally exhibit stability up to 45 - 55℃. pH Most intracellular enzymes exhibit optimal activity at pH values between 5 – 9. Extremes of pH can lead to enzyme denaturation. Michaelis-Menten Kinetics Michaelis-Menten Equation Vo = initial reaction velocity Vmax = maximal velocity = kcat [E]total Km = Michaelis constant = (k-1 + k2)/k1 [S] = substrate concentration Assumptions: 1. Substrate concentration is greater than enzyme concentration 2. Concentration of the ES complex does not change with time (steady state assumption) 3. Initial reaction velocities are used in the analysis of enzyme reactions.  Km = Michaelis constant; substrate concentration at which the reaction velocity is equal to ½ Vmax. It does not vary with enzyme concentration. High Km - low affinity Low Km - high affinity Km1 Km2  ZERO ORDER REACTION FIRST ORDER REACTION 1. [S] < Km = Velocity of reaction is approximately proportional to the substrate concentration 2. [S] > Km = Velocity of reaction is constant and equal to Vmax 3. Rate of reaction is directly proportional to enzyme concentration. Lineweaver Burk Plot or Double Reciprocal plot Lineweaver Burk Plot Straight line obtained after plotting reciprocal of Vo and [S]. Used to calculate Km and Vmax Used to determine mechanism of action of enzyme inhibitors Enzyme Inhibitors Substance that decreases the velocity of an enzyme-catalyzed reaction. Can be either reversible (noncovalent bonds) or irreversible (through covalent bonds). Competitive inhibition Inhibitor binds reversibly to the same site that the substrate would normally occupy. Competitive inhibition 1. Effect on Vmax: unchanged; increasing [S] will reach the Vmax observed in the absence of inhibitor 2. Effect on Km: increases Km. 3. Effect on Lineweaver-Burk Plot: inhibited and uninhibited reactions intersect on the y-axis at 1/Vmax (unchanged Vmax) and different x-intercepts at -1/Km (increased Km) HMG CoA reductase inhibitors are one of the examples of competitive inhibitor Competitive inhibition HMG CoA reductase inhibitors are one of the examples of a competitive inhibitor. Noncompetitive inhibition Occurs when the inhibitor and substrate bind at different sites on the enzyme. Noncompetitive inhibitor can bind either free enzyme or the enzyme- substrate complex – preventing the reaction from occurring. Noncompetitive inhibition Noncompetitive inhibition 1. Effect on Vmax: decrease apparent Vmax of the reaction. 2. Effect on Km: unchanged Km. 3. Effect on Lineweaver-Burk Plot: unchaged Km, decreased apparent Vmax. Oxypurinol, a metabolite of allopurinol, is a noncompetitive inhibitor of xanthine oxidase.  Please download and install the Slido app on all computers you use Which of the following is INCORRECT regarding a lineweaver-burk plot for a noncompetitive inhibitor? ⓘ Start presenting to display the poll results on this slide. Enzymes: Regulation Part 3 Allosteric Enzymes Effectors – bind noncovalently at a site other than the active site (allosteric site). Can be negative effectors (inhibitors) or positive effectors (increase enzyme activity) Allosteric Enzymes Homotropic effectors Substrates as effector Functions as a positive effector Sigmoidal curve Positive cooperativity Eg. Hemoglobin Allosteric Enzymes Heterotropic effectors Effectors different from substrate. Exemplified by feedback inhibition. Eg. Phosphofructokinase-1 is allosterically inhibited by citrate (not a substrate) Covalent Modification Phosphorylation and desphophorylation Phosphorylation – protein kinases – use ATP as phosphate donor Activates the enzyme Dephosphorylation – phosphoprotein phosphatase Deactivates the enzyme Covalent Modification Activation of zymogens Activation via proteolytic action of enteropeptidase (enterokinase) of trypsinogen → trypsin Trypsin catalyzes subsequent conversion of numerous other pancreatic zymogens Enzyme Synthesis Cells can also regulate the amount of enzyme present by altering the rate of enzyme synthesis. Increase or decrease of enzyme synthesis leads to alteration in the total population of active sites. Eg. Insulin production in response to hyperglycemia – induction of enzymes involved in glucose metabolism Mechanisms of Enzyme Regulation REGULATOR EVENT TYPICAL EFFECTOR RESULTS TIME REQUIRED FOR CHANGE Substrate availability Substrate Change in velocity Immediate Product inhibition Reaction Product Change in Vmax and/or Immediate Km Allosteric control Pathway end product Change in Vmax and/or Immediate K0.5 Covalent Modification Another enzyme Change in Vmax and/or Immediate to minutes Km Synthesis or Hormone or metabolite Change in the amount Hours to days degradation of enzyme of enzyme  Please download and install the Slido app on all computers you use Which of the following mechanisms of enzyme regulation usually takes hours to days for change? ⓘ Start presenting to display the poll results on this slide.  ENZYMES John Paul Martin D. Bagos, MD, FPCP, DPCEDM Assistant Professor, Department of Biochemistry and Nutrition Endocrinologist