Biochemistry Lecture Notes PDF - Bioenergetics & Metabolism

Biochemistry PHAR 284 I. Bioenergetics & Metabolism Lecture 2 Dr. Zeina Al Ariss Faculty of Pharmacy Beirut Arab University I. Bioenergetics & Metabolism Enzymes: Kinetics, inhibitors & regulatory enzymes Ga...

Biochemistry PHAR 284 I. Bioenergetics & Metabolism Lecture 2 Dr. Zeina Al Ariss Faculty of Pharmacy Beirut Arab University I. Bioenergetics & Metabolism Enzymes: Kinetics, inhibitors & regulatory enzymes Galal, A., Biochemistry for Pharmacy Students First Edition  Enzyme Kinetics During the formation of the reaction product, a high-energy intermediate or transition state intermediate (T*) is formed. The energy difference between reactants and T* is called the free energy of activation. In general, the lower the free energy of activation, the faster the rate of the reaction. Therefore, in order to increase the rate of biochemical reactions, enzymes catalyze these reactions by providing an alternate reaction pathway with a lower free energy of activation; this occurs without changing the free energies of reactants or products. Factors affecting reaction velocity 1. Substrate concentration The initial velocity of an enzyme-catalyzed reaction (Vo) increases with the increase in substrate concentration [S] until a maximal velocity (Vmax) is reached, where saturation of all available binding sites occurs (plateau). The substrate concentration at which Vo is half Vmax is known as Michaelis constant (Km). Km is characteristic of an enzyme and its particular substrate, and reflects the affinity of the enzyme for that substrate. However, Km does not vary with the concentration of enzyme. A small Km value reflects a high affinity of the enzyme for the substrate, because a low substrate concentration is needed to reach ½Vmax; however, a large Km value reflects a low affinity of enzyme for the substrate, because a high substrate concentration is needed to reach ½Vmax. If the reciprocal of Vo (1/vo) is plotted versus the reciprocal of [S] (1/[S]), a straight line (not hyperbolic) is obtained; this line is called the double-reciprocal plot or the Lineweaver-Burk plot. 2. Temperature The reaction velocity increases with temperature until a peak velocity is reached. Further elevation of the temperature results in a decrease in reaction velocity due to denaturation of the enzyme proteins. The optimum temperature for most human enzymes is between 35°C and 40°C. Human enzymes start to denature at temperatures above 40°C. 3. pH pH affects reaction velocity in 2 ways: a- the catalytic process usually requires that the enzyme and substrate have specific chemical groups in either ionized or un-ionized form in order to interact. b- extremes of pH can also lead to denaturation of the enzyme.  Enzyme Inhibitors Any substance that can diminish the velocity of an enzyme-catalyzed reaction is called an inhibitor. Irreversible inhibitors bind to enzymes through covalent bonds, while reversible inhibitors bind to enzymes through non-covalent bonds; reversible inhibition is further classified to competitive or non-competitive. Competitive enzyme inhibition: It occurs when the inhibitor competes with the substrate for the same site that it normally occupies by reversible binding to that site. In the presence of competitive inhibitors, Vmax is not affected because at high substrate concentration the effect of inhibitor can be reversed so that the reaction velocity can reach the Vmax observed in the absence of inhibitor. However, Km is apparently increased because more substrate is needed to reach Vmax. Non-Competitive enzyme inhibition: It occurs when the inhibitor and substrate bind to different sites on the enzyme. In this case, binding of the inhibitor does not affect binding of the substrate; however, the efficiency of the enzyme to act on the substrate is decreased. Non-competitive inhibition cannot be overcome by increasing substrate concentration; therefore, Vmax is apparently decreased. However, Km is the same in the presence or absence of the non-competitive inhibitor. Examples of drugs that act as enzyme inhibitors:  Captopril (antihypertensive) is a competitive inhibitor of the angiotensin- converting enzyme necessary for the conversion of angiotensin I to the potent vasoconstrictor, angiotensin II.  Methotrexate (anticancer) is a competitive inhibitor of dihydrofolate reductase necessary for the synthesis of folic acid, and subsequently DNA.  Atorvastatin (cholesterol-lowering) is a competitive inhibitor of HMG- CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis.  Efavirenz (antiviral) is a non-competitive inhibitor of reverse transcriptase necessary for viral replication.  Aspirin (anti-inflammatory) is an irreversible inhibitor of cyclooxygenase (COX) responsible for the biosynthesis of inflammatory mediators.  Regulatory Enzymes In metabolic pathways, enzymes work in multi-step reactions, where the reaction product of one enzyme becomes the substrate of the next enzyme. However, a given enzyme in each pathway has a greater effect in adjusting the rate of the overall reaction; this rate-limiting enzyme is known as a "regulatory" or “key” enzyme. The activity of these enzymes is regulated by: a. Allosteric modulation b. Covalent modification c. Alteration of enzyme synthesis a. Allosteric modulation Allosteric modulators (usually small metabolites or co-factors) bind reversibly to specific sites in regulatory enzymes, other than the active sites, known as allosteric sites. This binding produces conformational changes (i.e. change in shape) in the enzyme causing either an increase or a decrease in the Vmax or Km This is known as positive (+) or negative (-) modulation. Allosteric modulators may be either homotropic (i.e. the enzyme substrate itself is the modulator) or heterotropic (i.e. the modulator is a metabolite different from the enzyme substrate; e.g. feedback inhibition of the first enzyme in a metabolic pathway by the end-product). b. Covalent modification Many enzymes may be regulated by phosphorylation or dephosphorylation of OH group in amino acids in their active site; phosphorylation is catalyzed by the enzyme protein kinase using ATP as phosphate donor, while dephosphorylation is catalyzed by phosphoprotein phosphatase. Depending on the specific enzyme, the phosphorylated form may be more or less active than the unphosphorylated enzyme. c. Alteration of enzyme synthesis The regulatory mechanisms described above modify the activity of existing enzyme molecules. However, cells can also regulate the amount of enzyme present by altering the rate of enzyme synthesis (induction or repression) without affecting the activity of existing enzyme molecules. e.g. When the level of insulin increases as a result of high blood glucose levels, this induces the synthesis of key enzymes involved in glucose metabolism. This induction is slow (takes hours to days) compared with allosteric regulation in enzyme activity or covalent modification, which take seconds to minutes.  Isoenzymes Isoenzymes or isozymes are proteins that catalyze the same reaction, although, they have different structures due to different amino acid sequence. Isozymes have the following characteristics:  They have different charges and can be therefore separated by electrophoresis.  They are formed of different subunits in various combinations.  They differ in their kinetic and regulatory properties. Examples of isoenzymes include:  Glucokinase & hexokinase, which are isoenzymes that catalyze the phosphorylation of glucose in different organs (see glycolysis).  Creatine kinase, which exists as 3 isoenzymes formed by the combinations of 2 different subunits (B & M): 1. CK1 (BB), which is abundant in brain and smooth muscle (practically absent form serum) 2. CK2 (MB), which is abundant in cardiac muscle, some in skeletal muscle (practically absent from serum). 3. CK3 (MM), which is abundant in skeletal muscle and cardiac muscle (practically 100 % of serum CK) The pattern of isoenzymes found in plasma may, therefore, serve as a means of identifying the site of tissue damage. For example, the plasma levels of creatine kinase (CK) are commonly determined in the diagnosis of myocardial infarction via the appearance of CK2.  Enzymes of Clinical Diagnostic Value Certain enzymes are always present in the circulation to perform a given physiologic function (e.g. enzymes involved in blood clotting); these are known as "functional enzymes". Other enzymes, have no known physiologic function in blood (non-functional enzymes); they are normally present in blood as a result of normal cell turnover. Increase in the level of non-functional enzymes above normal reflects tissue damage resulting from disease. The following is a list of enzymes of clinical diagnostic value: I. Bioenergetics & Metabolism Oxidative decarboxylation of Pyruvate & The Krebs Cycle Galal, A., Biochemistry for Pharmacy Students First Edition  Bioenergetics & Role of ATP Bioenergetics is the study of energy changes that accompany biochemical reactions; it makes use of the field of thermodynamics to allow prediction of whether a biochemical reaction can take place. Free Energy The direction of a chemical reaction is determined by two factors: 1. Enthalpy (ΔH), which is a measure of the change in heat content of the reactants and products. 2. Entropy (ΔS), which is a measure of the change in randomness of reactants and products. When combined mathematically, enthalpy and entropy are used to determine the free energy change (G): G = H – T S (T is the absolute temperature in degrees Kelvin; K = C + 273) The sign of G is used to predict the direction of a reaction:  If ΔG is negative, the reaction proceeds spontaneously with a net loss of energy (reaction is said to be exergonic).  If ΔG is positive, the reaction proceeds only if energy is added to the system (reaction is said to be endergonic).  If ΔG = 0, the system is in equilibrium. In metabolic pathways, Gs of individual reactions are additives; this allows thermodynamically unfavorable (endergonic) reactions to proceed while coupled to highly exergonic reactions, provided that the overall pathway G is negative, as shown in the following example: The conversion of glucose to glucose 6-phosphate has a positive Go (Go is the standard free energy change, or G under standard temperature and pressure). (1) Glucose + Pi Glucose 6-phosphate + H2O Go = 13.8 KJ/mol Under normal physiological conditions, reaction (1) will not proceed unless coupled to a highly exergonic reaction, e.g. reaction (2). (2) ATP + H2O ADP + Pi Go = -30.5 KJ/mol Reactions (1) & (2) share common intermediates (H2O & Pi) and can be expressed by summing them up as follows: (1) + (2) Glucose + ATP Glucose 6-phosphate + ADP Go = -16.7 KJ/mol ATP as an Energy carrier ATP (adenosine triphosphate) plays a special role as the energy currency of the cell that links catabolism and anabolism. Cells obtain free energy in a chemical form by the catabolism of nutrients, and they use this energy to make ATP from ADP and inorganic phosphate (Pi). Then, ATP donates some of its chemical energy to endergonic processes such as:  synthesis of metabolic intermediates from smaller precursors.  transport of substances against concentration gradient.  muscle contraction. The bond shown as is termed a high- energy bond and the symbol indicates that the phosphate group attached to the bond, upon transfer to an appropriate acceptor, results in the transfer of a large quantity of free energy. Therefore, ATP has two high-energy bonds and ADP has one; however, AMP has a low-energy normal ester ATP link. Major sources of ATP: 1. Glycolysis 2. Citric acid cycle (Krebs cycle) 3. Respiratory chain  Cellular Respiration … an introduction to metabolism Cellular respiration is the group of molecular processes by which cells consume O2 and produce CO2; it occurs in three major stages: Stage 1: Organic fuel molecules (glucose, amino acids and fatty acids) are oxidized to yield acetyl-CoA (glucose produces pyruvate as intermediate, which is converted to acetyl-CoA through oxidative decarboxylation). Stage 2: Acetyl-CoA enters the citric acid cycle (Krebs cycle) to yield the reduced co-enzymes, NADH and FADH2. Stage 3: Reduced co-enzymes enter the respiratory chain, where O2 is reduced to H2O together with the production of ATP (oxidative phosphorylation). The following topics will be discussed in order: 1. Oxidative decarboxylation of pyruvate 2. Citric acid cycle 3. Respiratory chain This will be followed by the production of pyruvate from glucose, and acetyl- CoA from amino acids and fatty acids through detailed study of carbohydrates, proteins & lipids.  Oxidative Decarboxylation of Pyruvate Oxidative Decarboxylation of pyruvate is an irreversible process (i.e. highly favored thermodynamically) in which the -keto acid, pyruvate (end-product of aerobic glycolysis), is oxidized to acetyl-CoA in the matrix of mitochondria. This is catalyzed by a multi-enzyme complex known as pyruvate dehydrogenase complex. Pyruvate dehydrogenase complex is made up of 3 enzymes and 5 co- enzymes: The 3 enzymes are: E1: Pyruvate dehydrogenase E2: Dihydrolipoyl transacetylase E3: Dihydrolipoyl dehydrogenase The 5 co-enzymes are: 1. TPP (thiamine pyrophosphate) 2. Lipoate 3. Co-enzyme A (or CoA-SH) 4. FAD 5. NAD+ Regulation of pyruvate dehydrogenase complex Pyruvate dehydrogenase complex is the metabolic gateway between glycolysis & the citric acid cycle; it can be turned “ON” or “OFF” according to the metabolic state of the cell: 1. The enzyme complex is inhibited by its products, acetyl-CoA and NADH (negative feedback inhibition). 2. Phosphorylation of the enzyme complex decreases its activity while dephosphorylation increases its activity (covalent modification). Phosphorylation is initiated by increase in the following ratios: [Acetyl-CoA] / [CoA] or [NADH] / [NAD] or [ATP] / [ADP], i.e. signs of abundant energy supply. Clinical aspects 1. Arsenite and mercuric ions react with SH group in lipoate leading to inhibition of the enzyme complex and accumulation of pyruvate, which is converted to lactate causing fatal lactic acidosis. 2. Dietary deficiency of TPP inhibits the enzyme complex leading to fatal lactic acidosis. Generally, the brain is the most affected organ since it relies on glucose as fuel and is highly sensitive to acidosis; inhibition of the enzyme complex stops the conversion of glucose to acetyl-CoA with subsequent inhibition of energy production. N.B. Oxidative decarboxylation also occurs in the citric acid cycle (Krebs cycle), where -ketoglutarate is converted to succinyl-CoA by - ketoglutarate dehydrogenase complex.  Citric Acid Cycle (Krebs Cycle) The citric acid cycle, also known as tricarboxylic acid cycle (TCA), or Krebs cycle (after its discoverer, Hans Krebs), is the final aerobic pathway of oxidation of carbohydrates, proteins & lipids, whose common end-metabolite, acetyl-CoA, reacts with oxaloacetate to form citrate. By a series of dehydrogenation and decarboxylation, citrate is degraded releasing reduced co-enzymes (3 NADH + H+ & 1 FADH2) and 2 CO2 with the regeneration of oxaloacetate. Reduced co-enzymes are then oxidized in the respiratory chain with the formation of ATP (oxidative phosphorylation); thus, Krebs cycle is the major route for the generation of ATP and is located in the matrix of mitochondria. Krebs cycle has 8 steps: 1. Formation of citrate (6C) from the condensation of acetyl-CoA (2C) with oxaloacetate (4C) (catalyzed by citrate synthase). This reaction has a highly negative Go, which makes it irreversible; this is essential to operate the cycle because the concentration of oxaloacetate is very low (only a small quantity of oxaloacetate is needed for the oxidation of a large quantity of acetyl-CoA). N.B. CoA liberated from this reaction is recycled to participate in oxidative decarboxylation of another molecule of pyruvate. 2. Isomerization of citrate to isocitrate by 2 steps: dehydration to cis- aconitate, then rehydration to isocitrate (both catalyzed by aconitase). 3. Oxidative decarboxylation of isocitrate to -ketoglutarate (irreversible; catalyzed by isocitrate dehydrogenase); the 1st mol of NADH + H+ is produced. 4. Oxidative decarboxylation of -ketoglutarate to succinyl-CoA; (irreversible; catalyzed by -ketoglutarate dehydrogenase complex using the same 5 co-enzymes described with pyruvate dehydrogenase complex); the 2nd mol of NADH + H+ is produced. 5. Cleavage of the high-energy thioester bond of succinyl-CoA, with the formation of ATP or GTP (catalyzed by succinyl-CoA synthetase, also known as succinate thiokinase); this reaction is an example of substrate level phosphorylation.  6. Oxidation of succinate to fumarate (catalyzed by succinate dehydrogenase); one mol of FADH2 is produced (FAD, rather than NAD+, is involved because the reducing power of succinate is not sufficient to reduce NAD+). 7. Hydration of fumarate to malate (catalyzed by fumarase). 8. Oxidation of malate to regenerate oxaloacetate (catalyzed by malate dehydrogenase); the Go of this reaction is positive but it is driven by the highly exergonic reaction of citrate synthase; the 3rd and final mol of NADH + H+ is produced. Vitamins involved in the Krebs cycle: 1. Niacin 2. Riboflavin 3. Thiamine 4. Pantothenic acid Importance of the Krebs cycle: Krebs cycle is amphibolic; it serves in both catabolic and anabolic pathways; it is important for: 1. Production of ATP through the oxidation of acetyl-CoA resulting from carbohydrates, proteins and lipids. 2. Provision of the body with intermediate compounds essential for metabolism: a. Oxaloacetate is a precursor of the amino acid, aspartate, by transamination. b. -ketoglutarate is a precursor of the amino acid, glutamate, by transamination. c. Succinyl-CoA is essential for the synthesis of porphyrin ring of heme, which serves as oxygen carrier in hemoglobin. 3. Disposal of fumarate, which is a toxic metabolite produced in the urea cycle. Reactions of the Krebs cycle Inhibitors of the Krebs cycle: 1. Citrate synthase (step 1) is inhibited by its product, citrate, and by NADH and succinyl-CoA. 2. Aconitase (step 2) is inhibited by fluoroacetate (rodenticide): fluoroacetate is converted to fluoroacetyl-CoA condensing with oxaloacetate to form fluorocitrate (rather than citrate); fluorocitrate is a potent inhibitor of aconitase causing citrate to accumulate. 3. -ketoglutarate DH complex (step 4) is inhibited by arsenite and mercuric ions (see pyruvate dehydrogenase complex). 4. Succinate dehydrogenase (step 6) is inhibited by the succinic acid analog, malonic acid. Regulation of the Krebs cycle: The cycle is regulated by the enzymes that catalyze reactions with highly negative ΔG0: 1. Citrate synthase. 2. Isocitrate dehydrogenase. 3. α-ketoglutarate dehydrogenase complex. Energy yield of the Krebs cycle: During one turn of the cycle, 3 NADH and 1 FADH2 are produced. Oxidation of 1 NADH by the electron transport chain yields 3 ATP, whereas oxidation of 1 FADH2 yields 2 ATP. Totally, 12 ATP are produced per turn of the Krebs cycle.