Cmb Chapter 3

CHAPTER 3 - Bioenergetics, Enzymes, and Metabolism

Calculation of free energy changes •

BIOENERGETICS The study of the various types of energy

transformations that occur in living organisms. • Energy – capacity to do work, or the capacity to change or move something. • Thermodynamics – the study of the changes in energy that accompany events in the universe. The Laws of Thermodynamics 1. The first law of thermodynamics – the law of conservation of energy. − Energy can neither be created nor destroyed. − Transduction – conversion of energy from one to another. a. Cells are capable of energy transduction. b. Chemical energy is stored in certain biological molecules, such as ATP. c. Energy transduction in the biological world: conversion is the conversion of sunlight into chemical energy – photosynthesis.

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The universe can be divided into system and surroundings.  The system is a subset of the universe under study.  The surroundings are everything that is not part of the system.  The energy of the system is called the internal energy (E), and its change during a transformation is called ΔE. First law of thermodynamics: ΔE = Q – W, where E is the internal energy, Q is the heat energy and W is the work When there is energy transduction (ΔE) in a system, heat content may increase or decrease.  Reactions that lose heat are exothermic.  Reactions that gain heat are endothermic.  The first law does not predict whether an energy change will be positive or negative.

The second law of thermodynamics: events in the universe tend to proceed from a state of higher energy to a state of lower energy. − Such events are called spontaneous, they can occur without the input of external energy. −

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Non-standard conditions are corrected for prevailing conditions. – Equation: ΔG = ΔG°’ + RT ln Keq. – Prevailing conditions may cause ΔG to be negative, even when G°’ is positive. – Making ΔG negative may involve coupling endergonic and exergonic reactions in a sequence. – Simultaneously coupled reactions have a common intermediate. – ATP hydrolysis is often coupled to endergonic reactions in cells.

Equilibrium versus Steady-State Metabolism • • •

Cellular metabolism is non-equilibrium metabolism. Cells are open thermodynamic systems. Cellular metabolism exists in a steady state. – Concentrations of reactants and products remain constant, but not at equilibrium. – New substrates enter and products are removed. – Maintaining a steady state requires a constant input of energy, whereas maintaining equilibrium does not.

ENZYMES are catalysts that speed up chemical reactions. • •

Enzymes Enzymes – –

are almost always proteins. may be conjugated with non-protein components. Cofactors are inorganic enzyme conjugates. Coenzymes are organic enzyme conjugates.

Properties of Enzymes • • • • • •

Are present in cells in small amounts. Are not permanently altered during the course of a reaction. Cannot affect the thermodynamics of reactions, only the rates. Are highly specific for their particular reactants called substrates. Produce only appropriate metabolic products. Can be regulated to meet the needs of a cell.

Overcoming the Activation Energy Barrier • A small energy input, the activation energy (EA) is required for any chemical transformation. – –

The EA barrier slows the progress of thermodynamically unstable reactants. Reactant molecules that reach the peak of the EA barrier are in the transition state.

Loss of available energy during a process is the result of a tendency for randomness to increase whenever there is a transfer of energy.

Entropy is a measure of randomness or disorder. a. b. c. d. e.

Every event is accompanied by an increase in the entropy of the universe. Entropy associated with random movements of particles Living systems maintain a state of order, or low entropy It is energy not available to do additional work.

Loss of available energy equal TΔS, where ΔS is the change in entropy.

FREE ENERGY •

The first and second laws of thermodynamics can be combined and expressed mathematically.

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Equation: ΔH (enthalpy) = ΔG + TΔS

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Free energy, ΔG, is the energy available to do work. Spontaneity of the reaction is ΔG, if 0 it is endergonic. Spontaneity depends on both enthalpy and entropy.

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Free-Energy Changes in Chemical Reactions • • • •

All chemical reactions are theoretically reversible. All chemical reactions spontaneously proceed toward equilibrium (Keq = [C][D]/[A][B]). The rates of chemical reactions are proportional to the concentration of reactants. At equilibrium, the free energies of the products and reactants are equal (ΔG = 0). • Free energy changes of reactions are compared under standard conditions. • The standard free energy changes, ΔG°’, are described for each reaction under specific conditions. • Standard conditions are not representative of cellular conditions, but are useful to make comparisons. • Standard free energy changes are related to equilibrium: ΔG°’ = -RT ln K’eq

Enzymes lower the activation energy • Without an enzyme, only a few substrate molecules reach the transition state. • With a catalyst, a large proportion of substrate molecules can reach the transition state.

The Active Site portion of the enzyme substrate binds to − An enzyme interacts with its substrate to form an enzymesubstrate (ES) complex. − The active site and the substrate have complementary shapes that allow substrate specificity. Mechanisms of Enzyme Catalysis 1. Substrate Orientation: Multiple substrates brought together in correct orientation to catalyze reaction. 2. Changing Substrate Reactivity: substrate influenced by amino acid side chains at active site that alter chemical properties (e.g., charge) of substrate. • Changes in the reactivity of the substrate temporarily stabilize the transition state. – Acidic or basic R groups on the enzyme may change the charge of the substrate. – Charged R groups may attract the substrate. – Cofactors of the enzyme increase the reactivity of the substrate by removing or donating electrons. 3. Inducing Strain in the Substrate: enzyme changes conformation of substrate to bring closer to conformation of transition state. • Inducing strain in the substrate. – Shifts in the conformation after binding cause an induced fit between enzyme and the substrate. – Covalent bonds of the substrate are strained. 

Conformational changes and catalytic intermediates. Various changes in atomic and electronic structure occur in both the enzyme and substrate during a reaction. Using time-resolved crystallography, researchers have determined the three-dimensional structure of an enzyme at successive stages during a reaction

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Kinetics is the study of rates of enzymatic reactions under various experimental conditions. • Rates of enzymatic reactions increase with increasing substrate concentrations until the enzyme is saturated. –

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At saturation every enzyme s working at maximum capacity.

The velocity at saturation is called maximal velocity, Vmax. The turnover number is the number of substrate molecules converted to product per minute per enzyme molecule at Vmax.

The Michaelis constant (KM) is the substrate concentration at onehalf of Vmax. – Units of KM are concentration units. – The KM may reflect the affinity of the enzyme for the substrate. • •

Plots of the inverses of velocity versus substrate concentrations, such as the Lineweaver-Burk plot, facilitate estimating Vmax and KM. Temperature and pH can affect enzymatic reaction rates.

Enzyme inhibitors slow the rates for enzymatic reactions. • Irreversible inhibitors bind tightly to the enzyme. • Reversible inhibitors bind loosely to the enzyme. – Competitive inhibitors compete with the enzyme for active sites • Usually resemble the substrate in structure. • Can be overcome with high substrate/inhibitor ratios. – Noncompetitive inhibitors • Bind to sites other than active sites and inactivate the enzyme. • The maximum velocity of enzyme molecules cannot be reached. • Cannot be overcome with high substrate/inhibitor ratios.

The Growing Problem of Antibiotic Resistance •

Antibiotics target human metabolism without harming the human host. – Enzymes involved in the synthesis of the bacterial cell wall. – Components of the system by which bacteria duplicate, transcribe, and translate their genetic information. – Enzymes that catalyze metabolic reactions specific to bacteria.

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Antibiotics have been misused with dire consequences. – Susceptible cells are destroyed, leaving rare and resistant cells to survive and replicate. – Bacteria become resistant to antibiotics by acquiring genes from other bacteria by various mechanisms.

METABOLISM is the collection of bio-chemical reactions that occur

within a cell. • Metabolic pathways are sequences of chemical reactions. – Each reaction in the sequence is catalyzed by a specific enzyme. – Pathways are usually confined to specific locations. – Pathways convert substrates into end products via a series of metabolic intermediates. •

Catabolic pathways break down complex substrates into simple end products. – Provide raw materials for the cell. – Provide chemical energy for the cell.

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Anabolic pathways synthesize complex end products from simple substrates. – Require energy. – Use ATP and NADPH from catabolic pathways. •

Anabolic and catabolic pathways are interconnected. – In stage I, macromolecules are hydrolyzed into their building blocks. – In stage II, building blocks are further degraded into a few common metabolites. – In stage III, small molecular weight metabolites like acetyl-CoA are degraded yielding ATP.

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3-phosphoglycerate is converted to pyruvate via three sequential reactions, in one of them a kinase phosphorylates ADP. Glycolysis can generate a net of 2 ATPs for each glucose. Glycolysis occurs in the absence of oxygen, it is an anaerobic pathway. The end product, pyruvate, can enter aerobic or anaerobic catabolic pathways.

Anaerobic Oxidation of Pyruvate: The Fermentation Process Fermentation restores NAD+ from NADH. – – – – –

Oxidation-reduction (redox) reactions involve a change in the electronic state of reactants. – When a substrate gains electrons, it is reduced. – When a substrate loses electrons, it is oxidized. – When one substrate gains or loses electrons, another substance must donate or accept those electrons. • In a redox pair, the substrate that donates electrons is a reducing agent. • The substrate that gains electrons is an oxidizing agent. The Capture and Utilization of Energy • Reduced atoms can be oxidized, releasing energy to do work. • The more a substance is reduced, the more energy that can be released. • Glycolysis is the first stage in the catabolism of glucose, and occurs in the soluble portion of the cytoplasm. • The tricarboxylic (TCA) cycle is the second stage and it occurs in the mitochondria of eukaryotic cells. Glycolysis and ATP Formation • Of the reactions of glycolysis, all but three are near equilibrium (ΔG ~ 0) under cellular conditions. • The driving forces of glycolysis are these three reactions. • • • •

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Glucose is phosphorylated to glucose 6-phosphate by using ATP. Glucose 6-phosphate is isomerized to fructose 6-phosphate. Fructose 6-phosphate is phosphorylated to fructose 1,6-bisphophate using another ATP. Fructose 1,6-bisphosphate is split into two three-carbon phosphorylated compounds.

NAD+ is reduced to NADH when glyceraldehyde 3phosphate is converted to 1,3-bisphosphoglycerate. • Dehydrogenase enzymes oxidize and reduce cofactors. • NAD+ is a non protein cofactor associated with gluceraldehyde phosphate dehydrogenase. • NAD+ can undergo oxidation and reduction at different places in the cell. • NADH donates electrons to the electron transport chain in the mitochondria. ATP is formed when 1,3-bisphosphoglycerate is converted to 3-phosphoglycerate by 3-phosphoglycerate kinase. • Kinase enzymes transfer phosphate groups. • Substrate-level phosphorylation occurs when ATP is formed by a kinase enzyme. ATP formation is only moderately endergonic compared with other phosphate transfer in cells. • Transfer potential shown when molecules higher on the scale have less affinity for the group being transferred than are the ones lower on the scale. • The less the affinity, the better the donor.

Under anaerobic conditions, glycolysis depletes the supply of NAD+ by reducing it to NADH. In fermentation, NADH is oxidized to NAD+ by reducing pyruvate. In muscle and tumor cells pyruvate is reduced to lactate. In yeast and other microbes, pyruvate is reduced and converted to ethanol. Fermentation is inefficient with only about 8% of the energy of glucose captured as ATP.

Reducing Power • Anabolic pathways require a source of electrons to form larger molecules. • NADPH donates electrons to form large biomolecules. – NADPH is a non protein cofactor similar to NADH. – The supply of NADPH represents the cell’s reducing power. – NADP+ is formed by phosphate transfer from ATP to NAD+. • NADPH and NADH are interconvertible, but have different metabolic roles. • NADPH is oxidized in anabolic pathways. • NAD+ is reduced in catabolic pathways. • The enzyme transhydrogenase catalyzes the transfer of hydrogen atoms from one cofactor to the other. – NADPH is favored when energy is abundant. – NADH is used to make ATP when energy is scarce. Metabolic Regulation • Cellular activity is regulated as needed. • Regulation may involve controlling key enzymes of metabolic pathways. • Enzymes are controlled by alteration in active sites. – Covalent modification of enzymes regulated by phosphorylation such as protein kinases. – Allosteric modulation by enzymes regulated by compounds binding to allosteric sites. • In feedback inhibition, the product of the pathway allosterically inhibits one of the first enzymes of the pathway. Separating Catabolic and Anabolic Pathways • Glycolysis and gluconeogenesis are the catabolic and anabolic pathways of glucose metabolism. – Synthesis of fructose 1,6-bisphosphate is coupled to hydrolysis of ATP. – Breakdown of fructose 1,6-bisphosphate is via hydrolysis by fructose 1,6-bisphosphatase in gluconeogenesis. – Phosphofructokinase is regulated by feedback inhibition with ATP as the allosteric inhibitor. – Fructose 1,6-bisphosphatase is regulated by covalent modification using phosphate binding. – ATP levels are highly regulated. • Anabolic pathways do not proceed via the same reactions as the catabolic pathways even though they may have steps in common. – Some catabolic pathways are essentially irreversible due to large ΔG°’ values. – Irreversible steps in catabolic pathways are catalyzed by different enzymes from those in anabolic pathways.