Biochemistry Lecture 1.pdf

1. Foundation BTech-MTech Biotechnology Semester III BT 2013 Biochemistry  Biochemistry Biochemistry can be defined as the science concerned with the chemical basis of life (Gk bios “life”). The cell is the structural unit of living systems. Thus, bio...

1. Foundation BTech-MTech Biotechnology Semester III BT 2013 Biochemistry  Biochemistry Biochemistry can be defined as the science concerned with the chemical basis of life (Gk bios “life”). The cell is the structural unit of living systems. Thus, biochemistry can also be described as the science concerned with the chemical constituents of living cells and with the reactions and processes they undergo. By this definition, biochemistry encompasses large areas of cell biology, of molecular biology, and of molecular genetics. Because life depends on biochemical reactions, biochemistry has become the basic language of all biologic sciences. Biochemistry is concerned with the entire spectrum of life forms, from relatively simple viruses and bacteria to complex human beings. Metabolism  Metabolism Metabolic pathways are series of consecutive enzymatic reactions that produce specific products. Their reactants, intermediates, and products are referred to as metabolites. Each reaction in the metabolic pathways is catalyzed by a distinct enzyme, of which there are 4000 known. The reaction pathways often divided into two categories: 1. Catabolism, or degradation – nutrients and cell constituents are broken down exergonically to salvage their components and/or to generate free energy. 2. Anabolism, or biosynthesis – biomolecules are synthesized from simpler components. The free energy released by catabolic processes is conserved through the synthesis of ATP from ADP and phosphate or through the reduction of the coenzyme NADP to NADPH. ATP and NADPH are the major free energy sources for anabolic pathways. Energy relationships between catabolic and anabolic pathways  ATP and NADPH are the major free Overview of catabolism energy sources for anabolic pathways Catabolism: Large numbers of diverse substances (carbohydrates, lipids, and proteins) to common intermediates (mainly acetyl-CoA). These intermediates are then further metabolized in a central oxidative pathway that terminates in a few end products. Biosynthesis: Relatively few metabolites, mainly pyruvate, acetyl-CoA, and the citric acid cycle intermediates, serve as starting materials for a host of varied biosynthetic products.  Enzymes Biocatalyst - a biological substance that initiates or increases the rate of a chemical reaction in a living organisms without itself being affected: proteins (enzymes) in a few cases nucleic acids (ribozymes, Altman A and Cech TR, Nobel Price 1998)  Naming and classification of enzymes Enzymes names – traditionally end in “-ase” (Eg: amylase) – exceptions are proteolytic enzymes (Eg: Trypsin) Why classify enzymes? – Lack of consistency in the nomenclature ▪ Some names indicates the only the substrate; no nature of the reaction Eg: lactase and fumarase ▪ Some names explains only the nature of the reaction Eg: transcarboxylase ▪ Some enzymes make clear the substrate and nature of the reaction Eg: malate dehydrogenase ▪ Some enzymes known by more than one name International Union of Biochemistry appointed a commission to classify the enzymes in 1955 (Enzyme Commission). The first version was published in 1961. The current sixth edition, published in 1992, contains 3196 different enzymes.  The Enzyme Commission’s (EC) system of classification First Enzyme class Type of reaction catalyzed digit Oxidation/Reduction reactions 1 Oxidoreductases Transfer of H atoms, O atoms or electrons from one substrate to another Transfer of an atom or group between two molecules (excluding 2 Transferases reactions in other classes) AX + B ↔ BX + A Hydrolysis reactions 3 Hydrolases A-X + H2O ↔ X-OH + HA 4 Lyases Removal of a group from substrate (not by hydrolysis) 5 Isomerases Isomerization reactions The synthetic joining of two molecules, coupled with the breakdown of ATP or other NTPs. 6 Ligases X + Y + ATP ↔ X-Y + ADP + Pi X + Y + ATP ↔ X-Y + AMP + PPi The EC assigned to each enzyme (1) a code number of four elements separated by dots. First number : The main class Second number : The subclass Third number : The Sub-subclass Fourth number: The arbitrarily assigned serial number in sub-subclass (2) a systematic name - includes the name of the substrate or substrates and a word ending ‘-ase’ indicating the nature of the reaction. Example: Hexokinase Systematic name: ATP:glucose phosphotransferase – indicates catalyzes the transfer of a phosphoryl group from ATP to glucose. Classification number: EC 2.7.1.1. 2 : Transferase (main class) 7 : Phosphotransferase (subclass) 1 : Phosphotransferase with a hydroxyl group as acceptor (sub-subclass) 1 : Arbitrarily assigned serial number  Isoenzymes in EC classification In EC classification, isoenzymes (different enzymes catalyzing the same reactions) will have the same four number code. Eg: Five different isoenzymes of lactate dehydrogenase will have same identical code EC 1.1.1.27. Full EC classification can be found at: https://iubmb.qmul.ac.uk/  Enzyme synthesis and structure Enzyme synthesis and structure - Amino acids - Peptide bonds - Transcription - Translation - Post-translational modifications  Cofactors Enzymes catalyze a wide variety of chemical reactions. Their functional groups of polypeptide can facilely participate in acid–base reactions, form certain types of transient covalent bonds, and take part in charge–charge interactions. They are less suitable for catalyzing oxidation– reduction reactions and many types of group- transfer processes. Enzymes catalyze these reactions in association with small molecule cofactors, which essentially act as the enzymes’ “chemical teeth.”  Inorganic metal ions like Fe2+, Mg2+, or Mn2+ Cofactors Coenzymes – organic or metalloorganic molecule like biotin coenzyme A, PLP Prosthetic group: A coenzyme or metal ion that is very tightly or even covalently bound to the enzyme protein. Some enzymes require both a coenzyme and one or more metal ions for activity.  Many vitamins are coenzyme precursors Coenzyme Reaction Mediated Precursor in mammals Biotin Carboxylation Biotin (vitamin B7) Cobalamin (B12) coenzymes Alkylation Vitamin B12 Coenzyme A Acyl transfer Pantothenic acid (vitamin B5) Flavin coenzymes Oxidation-reduction Riboflavin (vitamin B2) Lipoic acid Acyl transfer Not required in diet Nicotinamide coenzymes Oxidation-reduction Nicotinic acid (niacin, Vit B3) Pyridoxal phosphate Amino group transfer Pyridoxine (vitamin B6) Tetrahydrofolate One-carbon group transfer Folate (vitamin B9) Thiamine pyrophosphate Aldehyde transfer Thiamine (vitamin B1)  The active site Binding sites link to specific groups in the substrate, ensuring that the enzyme and substrate molecules are held in a fixed orientation with respect to each other, with the reacting group or groups in the vicinity of catalytic sites. Catalytic site catalyzes the reaction. The region which contains the binding and catalytic site is termed the active site, or active centre, of the enzyme. Mammalian zinc Carboxypeptidase A metalloendopeptidase The active site - comprises only a small proportion of the total volume of the enzyme - is usually at or near the surface - it must be accessible to substrate molecules - in some cases, a clearly-defined pocket or cleft into which the whole or part of the substrate can fit - often includes both polar and non-polar amino acid residues, creating an arrangement of hydrophilic and hydrophobic microenvironments not found elsewhere on an enzyme molecule The binding and catalytic sites must be either amino acids or cofactors. Substrate binding involves a variety of weak non-covalent interactions. The amino acid residues in the active site which do not have a binding or catalytic function may nevertheless contribute to the specificity of the enzyme.  Specificity of enzyme action Enzymes are specific in action, exhibit both substrate and product specificity. Group specificity These enzymes act on several different, though closely related, substrates to catalyze a reaction involving a particular chemical group. Example: Alcohol dehydrogenase catalyzes the oxidation of a variety of alcohols. Hexokinase catalyzes the transfer of phosphate from ATP to several different hexose sugars. Absolute specificity These enzymes act only on one particular substrate. Example: Glucokinase catalyzes the transfer of phosphate from ATP to glucose and not to other sugar.  Stereochemical specificity If a substrate exist in two stereochemical forms, only one of the isomers will undergo reaction as a result of a catalysis by a particular enzyme. Example: L-amino acid oxidase mediates the oxidation of L-amino acids to oxo acids. D-amino acid oxidase mediates the oxidation of D- amino acids to oxo acids. The only enzymes which act on both stereoisomeric forms are those function is to interconvert L- and D- isomers. Eg: Alanine racemase which catalyzes: L-Alanine ↔ D-Alanine  Monomeric enzymes - Consist only a single polypeptide chain - Can not be dissociated into smaller units - Very few monomeric enzymes are known - All of these catalyze hydrolysis reactions - In general, they contain 100-300 amino acids (M.Wt. 13 – 35 kDa) - Some are associated with a metal ion (eg. Carboxypeptidase A) - Most are act without any cofactor. Examples: A number of proteases are monomeric enzymes (serine proteases, pepsin A, rennin, papain, exopeptidases like carboxy peptidase A and B). X-ray structure of bovine trypsin in covalent complex with its inhibitor leupeptin Other enzymes include ribonuclease and lysozyme.  Oligomeric enzymes Oligomeric enzymes consists of two or more polypeptide chains which are usually linked to each other by non-covalent interactions and never by peptide bonds. The component polypeptide chains are termed sub-units and may be identical to or different from each other. The molecular weight is usually in excess of 35 kDa. The vast majority of known enzymes are oligomeric enzymes. For example, all of the enzymes involved in glycolysis possess either two or four subunits. Lactate dehydrogenase  Free energy: The indicator of spontaneity Free energy: The amount of energy ‘free’ for work under the given conditions. J. Willard Gibbs defined the free energy content of any closed system as: G = H – TS H = Enthalpy, reflecting the number and kinds of bonds S = Entropy T = The absolute temperature (in Kelvin) When a chemical reaction occurs at constant temperature, the free-energy change, ΔG, is ΔG = ΔH – T ΔS A process tends to occur spontaneously only if ΔG is negative. The standard Gibbs free energy (ΔG0): Change of free energy that accompanies the formation of 1 mole of substance from its component elements, at their standard states (the most stable form of the element at 25°C and 100 kPa) The ΔG of a reaction varies with the total concentrations of its reactants and products and thus with their ionic states. So, ATP hydrolysis under physiological conditions has ΔG ≈ –50 kJ mol-1 rather than the –30.5 kJ mol-1 of its ΔG0.  Energy-coupling links the reactions in biology Cell function depends largely on molecules, such as proteins and nucleic acids, for which the free energy of formation is positive. To carry out these thermodynamically unfavorable, energy- requiring (endergonic) reactions, cells couple them to other reactions that liberate free energy (exergonic reactions), so that the overall process is exergonic: the sum of the free energy changes is negative. The coupling of exergonic and endergonic reactions by enzymes is absolutely central to the energy exchanges in living systems. 23 Enzyme kinetics  Chemical reactions The collision theory Molecules can react only if they come into contact with each other. Any factor which increases the collisions will increase the reaction rate. e.g. increased concentration of the reactants or increased temperature. However, not all colliding molecules will react, since, not all colliding molecules possess between them sufficient energy to undergo reaction. Activation energy and transition state theory Not all colliding molecules will react, since, not all colliding molecules possess between them sufficient energy to undergo reaction. The energy of an individual molecule will depend on, for example on what collisions that molecule has recently been involved in. In order for a reaction to take place, colliding molecules must have sufficient energy to overcome a potential-barrier known as the energy of activation. This is true even of energetically favorable reactions. The requirement of the activation energy is explained by transition state theory (Henry Eyring): Every chemical reaction proceeds via the formation of an unstable intermediate between reactants and products.  Catalysis A catalyst accelerates a chemical reaction without changing its extent and can be removed unchanged from amongst the end- products of the reaction. It has no overall thermodynamic effect: the amount of free energy liberated or taken up will be the same whether a catalyst is present or not. In most cases a catalyst acts by reducing the energy of activation. Uncatalyzed reaction: The catalyst combines with the reactants to form a different transition state of lower energy from that involved in the uncatalyzed reaction. Catalyzed reaction:  Kinetics of uncatalyzed chemical reactions The law of mass action (Guldberg and Waage, 1867) The rate of a reaction is proportional to the product of the active masses of each reactant, each active mass being raised to the power of the number of molecules of that reactant taking part. aA + bB → products Rate of the reaction v is proportional to [A]a x [B]b v = k [A]a x [B]b Order of reaction First order reaction If a reaction rate is proportional to the concentration of single reactant and the value of the exponent is one, then the reaction is said to be first order. Second-order reaction If a reaction rate is proportional to the concentration of two reactants or to the second power of a single reactant, then the reaction is said to be second order. Pseudo-first order reaction In second order reaction, if the concentration of a reactant remains constant (because it is a catalyst or it is in great excess with respect to the other reactants), then the reaction is said to be pseudo-first order. Zero-order reaction If a reaction rate is independent of the concentration of any of the reactants, then the reaction is said to be second order. Many enzyme-catalyzed reactions are zero-order when the reactant concentration is much greater than the enzyme concentration. Example: Detoxification of ethanol in the liver. Initial velocity (v0) The initial velocity (v0) of the reaction is the reaction rate at t = 0. v0 may be determined by drawing a tangent to the graph. Initial velocity will depend on the initial concentration of the reactants. V0 = k [A0]0 V0 = k [A0] V0 = k [A0]2  Kinetics of enzyme-catalyzed reactions: Introduction Wilhelmy (in 1850) showed acid-hydrolysis of sucrose is a pseudo-first-order reaction. Brown (1902) showed hydrolysis of sucrose by sucrase: - at low sucrose concentrations, the reaction was first-order, but - at higher concentrations it became zero-order. He proposed that the overall reaction is composed of two elementary Reactions: When the substrate concentration becomes high enough to entirely convert the enzyme to the ES form, the second step of the reaction becomes rate limiting and the overall reaction rate becomes insensitive to further increases in substrate concentration. The general expression for the velocity (rate) of this reaction is  The free energy profile of enzyme catalyzed reaction Relatively small changes in activation energy can greatly alter the rate of reaction: an enzyme which reduces the activation energy from 100 kJ mol-1 to 60 kJ mol-1 increases the rate by about 10 million. Enzyme-substrate complex: The enzyme initially binds the substrate at a specific binding-site to form relatively stable enzyme-substrate complex, this process takes place via the formation of an unstable transition state. In the enzyme-substrate complex, the reacting groups are held in close proximity to each other and to the catalytic-site of the enzyme. Transition state/Catalysis: The catalyzed reaction take place, via the formation of another unstable transition-state, to give the product. Enzyme-product complex: At this point, the product may still bound to the enzyme, the enzyme-product complex, would exist before the free product was liberated. Progress curves for the components of a simple single-substrate enzyme-catalyzed reactions  The Henri and Michaelis-Menten equations with Briggs and Haldane modifications Let us consider a single-substrate enzyme-catalyzed reaction where there is just only one substrate binding site per enzyme. (1) Early in the reaction, the concentration of the product, [P], is negligible, and the formation of ES from P can be ignored. This assumption simplifies the reaction to: (2) The general expression for the velocity (rate) of this reaction is (3) In order to be of use, kinetic expressions for overall reactions must be formulated in terms of experimentally measurable quantities. The quantities [ES] is not directly measurable. Rate of ES formation = k1 [E] [S] (4) Rate of ES breakdown = k-1 [ES] + k2 [ES] (5) At steady-state the rate of formation of ES is same as the breakdown of the ES. Therefore: (6) (7) (8) The term (k-1 + k2)/k1 is defined as the Michaelis constant, Km. The total enzyme present [E0] must be the sum of the concentration of free enzyme [E] and the bound enzyme [ES]. (9) (10) Substitute this in equation (8)  (11) (12) (13) (14) (15) (16) Substitute this [ES] derivation in rate equation (3) (17) The maximal velocity of a reaction, Vmax, occurs at high substrate concentrations when the enzyme is saturated, that is, when it is entirely in the ES form: Substitute Vmax in (17) (18) Usually the initial substrate concentration [S0] is very much greater than the initial enzyme concentration [E0]. So, the formation of ES will result in insignificant change in substrate concentration [S]. So, we can substitute [S0] for [S], giving: (19) When V0 is ½ V max Km is the value of [S0] which gives the initial velocity equal to ½ Vmax. Km will have the same unit as [S].  The significance of the Michaelis-Menten equation 1. Michaelis Constant (Km) Km is the substrate concentration at which the reaction velocity is half-maximal. Therefore, if an enzyme has a small value of Km , it achieves maximal catalytic efficiency at low substrate concentrations. Km is therefore also a measure of the affinity of the enzyme for its substrate as Km decreases, the enzyme’s affinity for substrate increases.  2. Turnover number (kcat) Turnover number (kcat) is obtained from the general expression: Vmax = kcat [E0] Vmax kcat = [E0] For enzymes with simple single substrate reactions kcat = k2 For enzymes with complicated mechanisms, kcat will be a function of several rate constants. Turnover number of an enzyme (kcat) is defined as the maximum number of reaction processes (turnovers) that each active site catalyzes per unit time. 3. Catalytic efficiency (Kcat/Km) The quantity kcat/Km is a measure of an enzyme’s catalytic efficiency. kcat Catalytic efficiency = Km  The Lineweaver-Burk plot The graph of the Michaelis-Menten equation, v0 against [S0], is unsatisfactory as a means of determining Vmax and Km. Hence, it is very difficult to use this plot to obtain an accurate value of Vmax and hence Km. Lineweaver and Burk (1934) overcame this problem by simply took the Michaelis-Menten equation, and inverted it: Michaelis-Menten equation: Inverted MM equation: Lineweaver-Burk equation: Lineweaver and Burk equation: Simple linear equation: y = mx + c m = slope c = y intercept  Enzyme Units and Specific Activity 1 unit (U) is the amount of enzyme that catalyses the reaction of specific amount of (eg: 1 nmol) of substrate per minute under standard or specified conditions. SI unit is katal (kat) which is defined as the amount of enzyme that which catalysees the transformation of one mole of substance per second. Specific activity: Activity of an enzyme per milligram of total protein (expressed in units/mg).  Problems 1. Given the reaction of an enzyme that follows Michaelis-Menten kinetics: k1 k2 E + S ↔ ES → E + P k-1 If Km = 30 mM and Vmax = 60 µmol min-1 a) What is the v0 at a substrate concentration of 0.1 mM? b) What is the v0 at a substrate concentration of 30 mM? c) What is the v0 at a substrate concentration of 1000 mM? 2. An enzyme catalyzed reaction exhibits a rate of 220 μmol/min at a substrate concentration of 28 mM. The Vmax is 250 μmol/min. What is v when [S] = 5 mM ? 3. Use the Michaelis-Menton Equation to calculate the missing values of [S] ([S]1 to [S]4) given below if Vmax = 5 mmol/min. [S] (mM) V (mmol/min) _______ ___________ 10 1.2 [S]1 1.7 [S]2 2.1 [S]3 2.2 [S]4 2.5 4. The kinetics of an enzyme has been measured as a function of substrate concentration (see Table) and plotted the data in Lineweaver-Burk plot (Figure below). For these assays, 1 nmol of enzyme was used. From these data, determine Vmax, Km, kcat, and the catalytic efficiency for the enzyme.  Pepsin Effects of pH on enzyme activity Optimum pH : 1.6 Enzymes are pH sensitive and have an optimum pH (or pH range) at Gastric juice pH : 1-2 which their activity is maximal; activity decreases at higher or lower pH. The pH optimum for the activity of an enzyme is generally close to the pH of the environment in which the enzyme is normally found. The effects of pH is a result of a combination of following factors: (1) the binding of substrate to enzyme, Glucose 6-phosphatase (2) the catalytic activity of the enzyme, Optimum pH : 7.8 Hepatocyte cytosol pH : 7.2 (3) the ionization of substrate, and (4) the variation of protein structure like oligomerization, denaturation, precipitation) (usually significant only at extremes of pH).  Effects of ionic strength on enzyme activity Ionic strength: half of total sum of concentration (ci) of every ionic species (i) in the solution times the square of its charge (zi). I = 0.5∑(cizi2) Example: ionic strength of 0.1 M CaCl2 is I = 0.5 x ((0.1 x 22) + (0.2 x 12)) = 0.3 M Effects on enzyme catalysis Ionic strength influence the movement of charged molecules such as (a) binding of charged substrate to enzyme (b) movement of charged groups within active site Effects on enzyme structure Ionic strength affects the enzyme structure by salting in/out  Effects of temperature on enzyme activity ❖ Effects on enzyme structure High temperature causes irreversible denaturation (unfolding) and structural changes in active site ❖ Effects on enzyme catalysis catalytic rate generally increase with temperature Low temp High temp  Enzyme cooperativity Ligand: Anything which binds to an enzyme or other protein is a ligand, regardless whether or not it is a substrate and undergoes a subsequent reaction. If more than one ligand-binding site is present on a protein, there is a possibility of interaction between the binding sites during the binding process. This is termed cooperativity. No coperativity Coperativity  Allosteric regulation In addition to active sites, allosteric enzymes generally have one or more regulatory, or allosteric sites for binding the modulator. Each regulatory site is specific for its modulator. Allosteric enzymes are generally larger and more complex than non-allosteric enzymes. Most have two or more subunits. Activator binding increases the concentration of the substrate-binding R state. The presence of activator therefore increases the protein’s substrate-binding affinity. The presence of inhibitor reduces the binding affinity for substrate by increasing the concentration of the T state.  Allosteric feedback inhibition In some multienzyme systems, the regulatory enzyme is specifically inhibited by the end product of the pathway whenever the concentration of the end product exceeds the cell’s requirements. The rate of production of the pathway’s end product is thereby brought into balance with the cell’s needs. This type of regulation is called feedback inhibition.  Characteristics of metabolic pathways 1. Metabolic pathways are irreversible A highly exergonic reaction is irreversible; that is, it goes to completion. If such a reaction is part of a multistep pathway, it confers directionality on the pathway; that is, it makes the entire pathway irreversible. 2. Catabolic and anabolic pathways must differ If two metabolites are metabolically interconvertible, the pathway from the first to the second must differ from the pathway from the second back to the first. The existence of independent interconversion routes, as we shall see, is an important property of metabolic pathways because it allows independent control of the two processes. 3. Every metabolic pathway has a first committed step Although metabolic pathways are irreversible, most of their component reactions function close to equilibrium. Early in each pathway, however, there is an irreversible (exergonic) reaction that “commits” the intermediate it produces to continue down the pathway. 4. All metabolic pathways are regulated Metabolic pathways are regulated by laws of supply and demand. Most metabolic pathways are controlled by regulating the enzymes that catalyze their first committed step(s); it prevents the unnecessary synthesis of metabolites further along the pathway. 5. Metabolic pathways in eukaryotic cells occur in specific cellular locations The compartmentation of the eukaryotic cell allows different metabolic pathways to operate in different locations. In multicellular organisms, compartmentation is carried a step further to the level of tissues and organs. For example, 1. The liver is largely responsible for the gluconeogenesis so as to maintain a relatively constant level of glucose in the circulation. 2. Adipose tissue is specialized for the storage and mobilization of triacylglycerols.  Phosphate compounds - ATP The endergonic processes that maintain the living state are driven by the exergonic reactions of nutrient oxidation. This coupling is most often mediated through the syntheses of a few types of “high-energy” intermediates whose exergonic consumption drives endergonic processes. These intermediates therefore form a sort of universal free energy “currency” through which free energy consuming processes in biological systems. Adenosine triphosphate (ATP), which occurs in all known life-forms, is the “high-energy” intermediate that constitutes the most common cellular energy currency. Phosphoryl-transfer reactions are of enormous metabolic significance  The role of ATP in phosphoryl-transfer reactions ΔG of phosphate hydrolysis Phosphate group-transfer potential – negatives of ΔG of phosphate hydrolysis –measure of the tendency of phosphorylated compounds to transfer their phosphoryl groups to water. ATP has an intermediate phosphate group-transfer potential; it serve as an energy conduit between “high- energy” phosphate donors and “low-energy” phosphate acceptors. In cells, kinases catalyze the transfer of phosphoryl groups between ATP and other molecules. Eg: pyruvare kinase, creatine kinase, hexokinase. ΔG of ATP hydrolysis varies with pH, divalent metal ion concentration, and ionic strength ΔG’0 = The standard Gibbs free energy (change of free energy that accompanies the formation of 1 mole of substance from its component elements, at their standard states (the most stable form of the element at 25°C and 100 kPa) Gas constant, R = 8.315 J/mol.K K’eq = Standard equilibrium constant ΔG’0 of ATP hydrolysis is –30.5 kJ mol-1 The ΔG of hydrolysis of phosphorylated compounds (like ATP) are highly dependent on the total concentrations of reactants and products, pH, divalent metal ion concentration (divalent metal ions such as Mg2+ have high phosphate-binding affinities), and ionic strength. Reasonable estimates of these quantities indicate that ATP hydrolysis under physiological conditions has ΔG ≈ –50 kJ mol-1 rather than the –30.5 kJ mol-1 of its ΔG0. It is important to keep in mind that 1. Within a given cell, the concentrations of most substances vary both with location and time. 2. The concentrations of many ions, coenzymes, and metabolites commonly vary by several orders of magnitude across membranous organelle boundaries. Unfortunately, it is usually quite difficult to obtain an accurate measurement of the concentration of any particular chemical species in a specific cellular compartment. Calculate the actual free energy of hydrolysis of ATP, ΔGp, in human erythrocytes. The standard free energy of hydrolysis of ATP is -30.5 kJ/mol, and the concentrations of ATP, ADP, and Pi in erythrocytes are as shown in the Table. Assume that the pH is 7.0 and the temperature is 37 °C (body temperature).  Formation of ATP 1. Substrate-level phosphorylation ATP may be formed from phosphoenolpyruvate by direct transfer of a phosphoryl group to ADP. Such reactions, which are referred to as substrate-level phosphorylations, most commonly occur in the early stages of carbohydrate metabolism. 2. Oxidative phosphorylation and photophosphorylation Both oxidative metabolism and photosynthesis act to generate a proton (H+) concentration gradient across a membrane. Discharge of this gradient is enzymatically coupled to the formation of ATP from ADP and Pi. In oxidative metabolism, this process is called oxidative phosphorylation, whereas in photosynthesis it is termed photophosphorylation. Most of the ATP produced by respiring and photosynthesizing organisms is generated in this manner.  Consumption of ATP 1. Early stages of nutrient breakdown – Example: early reactions in glycolysis 2. Metabolic reactions and Physiological processes The hydrolysis of ATP to ADP and Pi energizes many essential endergonic reactions as well as endergonic physiological processes such as chaperone-assisted protein folding, muscle contraction, and the transport of molecules and ions against concentration gradients. 3. Interconversion of nucleoside triphosphates Many biosynthetic processes, such as the synthesis of proteins and nucleic acids, require nucleoside triphosphates (NTPs) (ribonucleoside and deoxyribonucleoside triphosphates), other than ATP. All these NTPs are synthesized from ATP and the corresponding nucleoside diphosphate (NDP). 4. Additional phosphoanhydride cleavage in highly endergonic reactions Although many reactions involving ATP yield ADP and Pi, some reactions yield AMP and PPi. In these latter cases, the released PPi is rapidly hydrolyzed to 2Pi by inorganic pyrophosphatase. Examples: 1. The attachment of amino acids to tRNA molecules for protein synthesis. 2. Nucleic acid biosynthesis from the appropriate NTPs releases PPi.  Rate of ATP turnover The amount of ATP in a cell is typically only enough to supply its free energy needs for a minute or two. Hence, ATP is continually being hydrolyzed and regenerated; ATP act as a free energy transmitter rather than a free energy reservoir. Indeed, the metabolic half-life of an ATP molecule varies from seconds to minutes depending on the cell type and its metabolic activity. For instance, brain cells have only a few seconds’ supply of ATP (which, in part, accounts for the rapid deterioration of brain tissue by oxygen deprivation). An average person at rest consumes and regenerates ATP at a rate of 3 mol (1.5 kg) h–1 and as much as an order of magnitude faster during strenuous activity.  Phosphocreatine provides a “high-energy” reservoir for ATP formation Muscle and nerve cells (have a high ATP turnover) have a free energy reservoir (phosphocreatine in vertebrates) that functions to regenerate ATP rapidly. The above reaction is endergonic under standard conditions. However, the intracellular concentrations of its reactants and products (typically 4 mM ATP and 0.013 mM ADP) are such that it operates close to equilibrium (ΔG ≈ 0). When the cell is in a resting state, so that [ATP] is relatively high, the reaction proceeds with net synthesis of phosphocreatine, whereas at times of high metabolic activity, when [ATP] is low, the equilibrium shifts so as to yield net synthesis of ATP. A resting vertebrate skeletal muscle normally has sufficient phosphocreatine to supply its free energy needs for several minutes (but for only a few seconds at maximum exertion). In the muscles of some invertebrates, such as lobsters, phosphoarginine performs the same function.  Biological Oxidation-Reduction Reactions Oxidation–reduction reactions: - Reactions involving the transfer of electrons - living things derive most of their free energy from them. Photosynthesis : CO2 is reduced (gains electrons) and H2O is oxidized (loses electrons) to yield carbohydrates and O2 that is powered by light energy. Aerobic metabolism : The overall photosynthetic reaction is reversed so as to harvest the free energy of oxidation of carbohydrates and other organic nutrients. Every time we use a motor, an electric light or heater, or a spark to ignite gasoline in a car engine, we use the flow of electrons to accomplish work. Living cells have an analogous biological “circuit,” with a relatively reduced compound such as glucose as the source of electrons. As glucose is enzymatically oxidized, the released electrons flow spontaneously through a series of electron-carrier intermediates to another chemical species, such as O2. This electron flow is exergonic, because O2 has a higher affinity for electrons than do the electron-carrier intermediates. The resulting emf (electromotive force, the force proportional to the difference in electron affinity) provides energy to a variety of molecular energy transducers (enzymes and other proteins) that do biological work. Example of Redox reaction: Electrochemical cell Electron donor or reductant or reducing agent: Cu+ Electron acceptor or oxidant or oxidizing agent: Fe3+ Redox reactions may be divided into two half- reactions or redox couples, such as Conjugate redox pair: the electron donor and its conjugate electron acceptor: The pair of Cu+ and Cu2+ The two half-reactions of a redox reaction, each consisting of a conjugate redox pair, may be physically separated so as to form an electrochemical cell. Standard reduction potential, Eo: The relative affinity of the electron acceptor of each redox pair for electrons. It is measured as the potential difference when the conjugate redox pair, at 1 M concentrations, 25 °C, and pH 7, is connected with the standard (pH 0) hydrogen electrode. Eo = 0 A half-cell that takes electrons from the standard hydrogen cell is assigned a positive value of Eo, and one that donates electrons to the hydrogen cell, a negative value. The actual reduction potential, (Nernst equation) ΔEo = Eo of electron acceptor - Eo of electron donor ℱ = 96,480 J/V. mol (Faraday constant) Standard Reduction Potentials of Some Biologically Important Half-Reactions  Calculation of ΔG for redox reactions (a) Calculate the standard free-energy change, ΔGo, for (a) the following reaction: ΔEo = Eo of e- acceptor - Eo of e- donor Acetaldehyde + NADH → ethanol + NAD+ = –0.197 – (-0.320) = 0.123 V The relevant half-reactions and their Eo values are: ΔGo = – n ℱ ΔEo = – 2 x 96500 J/V.mol x 0.123 V (1) Acetaldehyde + 2H+ + 2e– → ethanol Eo = –0.197 V (2) NAD+ + 2H+ + 2e – → NADH + H+ Eo = –0.320 V = – 23,700 J/mol = – 23.7 KJ/mol (b) (b) Then calculate the actual free-energy change, ΔG, + ethanol [NAD ] when [acetaldehyde] and [NADH] are 1.00 M, and ΔG = ΔGo + RT ln acetaldehyde [NADH] [ethanol] and [NAD+] are 0.100 M. 0.1 x 0.1 ΔG = –23700 + (8.315 x 298 x ln ) 1x1 ℱ = ℱ = 96,480 J/V.mol R = 8.315 J/mol.K 0.1 x 0.1 ΔG = –23700 + (2.488.315 x 298 x ln ) 1x1 Δ𝐺 = –35100 J/mol = –35.1 KJ/mol  Metabolic regulation and control Why metabolic flow must be controlled? ✓ To provide products at the rate they are needed (to balance supply with demand). ✓ To maintain the steady-state concentrations of the intermediates in a pathway within a narrow range (homeostasis). Why organisms must maintain homeostasis? ✓ In an open system, such as metabolism, the steady state is the state of maximum thermodynamic efficiency. ✓ Many intermediates participate in more than one pathway, so that changing their concentrations may disturb a delicate balance. ✓ The rate at which a pathway can respond to a control signal slows if large changes in intermediate concentrations are involved. ✓ Large changes in intermediate concentrations may have deleterious effects on cellular osmotic properties. 71 Description of metabolic control and regulation: Every metabolic pathway has a rate-limiting step and is regulated by controlling the rate of this pivotal enzyme. These so-called regulatory enzymes are almost invariably allosteric enzymes subject to feedback inhibition and are often also controlled by covalent modification. Are these regulatory enzymes really rate limiting for the pathway? Is there really only one step in the pathway that is rate limiting?, or might there be a number of enzymes contributing to the regulation of the pathway? Does controlling these enzymes really control the flux of metabolites through the pathway?, or is the function of feedback inhibition really to maintain a steady state? 72  Mechanisms of metabolic flux control (or) Mechanisms of the control of regulatory enzymes The flux through a rate-determining step(s) of a pathway may be altered by several mechanisms: 1. Allosteric control Many enzymes are allosterically controlled by effectors that are often substrates, products, or coenzymes in the pathway. 2. Covalent modification (enzymatic interconversion) Many enzymes that regulate pathway fluxes have specific sites that may be enzymatically phosphorylated and dephosphorylated at specific Ser, Thr, and/or Tyr residues or covalently modified in some other way. Such enzymatic modification processes, which are themselves subject to control, greatly alter the activities of the modified enzymes. 3. Substrate cycles The existence of a substrate cycle (opposite reactions catalyzed by different enzymes) increases sensitivity of control than the flux through a single unopposed nonequilibrium reaction. 4. Genetic control Enzyme concentrations, and hence enzyme activities, may be altered by protein synthesis in response to metabolic needs. Mechanisms 1 to 3 can respond rapidly (within seconds or minutes) to external stimuli and are therefore classified as “short-term” control mechanisms. Mechanism 4 responds more slowly to changing conditions (within hours or days in higher organisms) and is therefore referred to as a “long-term” control mechanism.