Bioenergetics, Enzymes, and
Metabolism

• The study of the various types of energy transformations that occur in living organisms.

The Laws of Thermodynamics and the Concept of Entropy
• 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 First Law 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 form to another.
• Electric energy can be transduced to mechanical energy when we plug in a clock.

The First Law of Thermodynamics
(2)
• Cells are capable of energy transduction.
• Chemical energy is stored in certain biological molecules, such as ATP.
• Chemical energy is converted to mechanical energy when heat is released during muscle contraction.

The First Law of Thermodynamics
(3)
• Energy transduction in the biological world: conversion is the conversion of sunlight into chemical energy
– photosynthesis.
• Animals, such as fireflies and luminous fish, are able to convert chemical energy back to light.

The First Law of Thermodynamics
(4)
• 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 .

The First Law of Thermodynamics
(5)
• The first law of thermodynamics: ΔE = Q – W, where Q is the heat energy and W is the work energy.
• 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 First Law of Thermodynamics
(6)

The Second Law of
Thermodynamics (1)
• 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 .
– Loss of available energy during a process is the result of a tendency for randomness to increase whenever there is a transfer of energy.

The Second Law of
Thermodynamics (2)
• Entropy is a measure of randomness or disorder.
– It is energy not available to do additional work.
– Loss of available energy equal T ΔS , where
ΔS is the change in entropy.

The Second Law of
Thermodynamics (3)
• Every event is accompanied by an increase in the entropy of the universe.
– Entropy associated with random movements of particles or matter.
– Living systems maintain a state of order, or low entropy

• The first and second laws of thermodynamics can be combined and expressed mathematically.
– Equation: ΔH = ΔG + TΔS
– Free energy, ΔG, is the energy available to do work.
– Spontaneity of the reaction is ΔG, if <0 the reaction is exergonic, if >0 it is endergonic.
– Spontaneity depends on both enthalpy and entropy.

Free-Energy Changes in Chemical
Reactions (1)
• All chemical reactions are theoretically reversible.
• All chemical reactions spontaneously proceed toward equilibrium (K eq
= [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 in Chemical
Reactions (2)
• 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

Calculation of free energy changes
(1)
• Non-standard conditions are corrected for prevailing conditions.
– Equation: ΔG = ΔG°’ + RT ln K eq
.
– 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.

Calculation of free energy changes
(1)

Coupling Endergonic and
Exergonic Reactions

Coupling Endergonic and
Exergonic Reactions

Equilibrium versus Steady-State
Metabolism
• Cellular metabolism is nonequilibrium 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.

3.2 Enzymes as Biological
Catalysts
• Enzymes are catalysts that speed up chemical reactions.
• Enzymes are almost always proteins.
• Enzymes may be conjugated with nonprotein components.
– Cofactors are inorganic enzyme conjugates.
– Coenzymes are organic enzyme conjugates.

• 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
(E
A
) is required for any chemical transformation.
– The E
A barrier slows the progress of thermodynamically unstable reactants.
– Reactant molecules that reach the peak of the EA barrier are in the transition state .

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.

• An enzyme interacts with its substrate to form an enzyme-substrate (ES) complex .
• The substrate binds to a portion of the enzyme called the active site .
• The active site and the substrate have complementary shapes that allow substrate specificity.

Mechanisms of Enzyme Catalysis
(1)
• Substrate orientation means enzymes hold substrates in the optimal position of the reaction.

Mechanisms of Enzyme Catalysis
(2)
• 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.

Mechanisms of Enzyme Catalysis
(3)
• 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.

Mechanisms of Enzyme Catalysis
(4)
• 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 threedimensional structure of an enzyme at successive stages during a reaction

Mechanisms of Enzyme Catalysis
(4)
• 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 threedimensional structure of an enzyme at successive stages during a reaction

• 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.
– At saturation every enzyme s working at maximum capacity.
– The velocity at saturation is called maximal velocity ,
V max
.
– The turnover number is the number of substrate molecules converted to product per minute per enzyme molecule at V max
.

• The Michaelis constant (K
M
) is the substrate concentration at onehalf of Vmax.
– Units of K
M are concentration units.
– The K
M 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
V max and K
M
.
• 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 Human Perspective: The
Growing Problem of Antibiotic
Resistance (1)
• 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.

The Human Perspective: The
Growing Problem of Antibiotic
Resistance (2)
• 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.
• 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.

Oxidation and Reduction: A Matter of Electrons (1)
• 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 .

Oxidation and Reduction: A Matter of Electrons (1)

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 (1)
• 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.

Glycolysis and ATP Formation (2)

Glycolysis and ATP Formation (3)
• Glucose is phosphorylated to glucose 6phosphate 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.

Glycolysis and ATP Formation (4)
• NAD + is reduced to NADH when glyceraldehyde
3-phosphate is converted to 1,3bisphosphoglycerate.
– Dehydrogenase enzymes oxidize and reduce cofactors.
– NAD + is a nonprotein 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.

Glycolysis and ATP Formation (5)

Glycolysis and ATP Formation (5)

Glycolysis and ATP Formation (6)
• ATP is formed when 1,3bisphosphoglycerate is converted to 3phosphoglycerate by 3phosphoglycerate kinase.
– Kinase enzymes transfer phosphate groups.
– Substrate-level phosphorylation occurs when ATP is formed by a kinase enzyme.

Glycolysis and ATP Formation (7)
• 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.

Glycolysis and ATP Formation (8)
• 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 Process of Fermentation (1)
• Fermentation restores NAD + from NADH.
– 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.

Anaerobic Oxidation of Pyruvate:
The Process of Fermentation (2)

• Anabolic pathways require a source of electrons to form larger molecules.
• NADPH donates electrons to form large biomolecules.
– NADPH is a nonprotein 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.

• 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 (1)
• 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 .

Separating Catabolic and Anabolic
Pathways (2)

Separating Catabolic and Anabolic
Pathways (3)
• 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.