8 Energy, Enzymes, and Metabolism. 8 Energy, Enzymes, and Metabolism 8.1 What Physical Principles Underlie Biological Energy Transformations? 8.2 What

8Energy, Enzymes, and

Metabolism

8 Energy, Enzymes, and Metabolism

8.1 What Physical Principles Underlie Biological Energy Transformations?

8.2 What Is the Role of ATP in Biochemical Energetics?

8.3 What Are Enzymes?

8.4 How Do Enzymes Work?

8.5 How Are Enzyme Activities Regulated?

8 Energy, Enzymes, and Metabolism

Opening Question:

How are enzymes used in other industrial processes?

Many laundry aids have been developed that include various enzymes to hydrolyze proteins, fats, and starches to remove a variety of stains.


A chemical reaction occurs when atoms have enough energy to combine or change bonding partners.

sucrose + H2O → glucose + fructose

reactants products


Metabolism: the sum total of all chemical reactions occurring in a biological system at a given time.

Metabolic reactions involve energy changes.


Energy is the capacity to do work, or the capacity for change.

In biochemical reactions, energy changes are associated with changes in the composition and properties of molecules.


All forms of energy are either:

• Potential energy—energy stored as chemical bonds, concentration gradient, charge imbalance, etc.

• Kinetic energy—the energy of movement.

Energy can be converted from one form to another.

Figure 8.1 Energy Conversions and Work

Table 8.1


Two types of metabolism:

• Anabolic reactions: complex molecules are made from simple molecules, and energy input is required.

• Catabolic reactions: complex molecules are broken down to simpler ones, and energy is released.


Catabolic and anabolic reactions are often linked.

The energy released in catabolic reactions is used to drive anabolic reactions—to do biological work.


The laws of thermodynamics apply to all matter and all energy transformations in the universe.

They help us to understand how cells harvest and transform energy to sustain life.


First law of thermodynamics: energy is neither created nor destroyed.

When energy is converted from one form to another, the total energy before and after the conversion is the same.


Second law of thermodynamics: when energy is converted from one form to another, some of that energy becomes unavailable to do work.

No energy transformation is 100 percent efficient; some energy is lost to disorder.

Figure 8.2 The Laws of Thermodynamics


Entropy is a measure of the disorder in a system.

It takes energy to impose order on a system. Unless energy is applied to a system, it will be randomly arranged or disordered.


In any system:

Total energy = usable energy + unusable energy

H = G + TS

enthalpy (H) = free energy (G) + entropy (S)

G = H – TS

(T = absolute temperature)


Free energy (G) is the usable energy that can do work.

Change in energy can be measured in calories or joules.

Change in free energy (ΔG) in a reaction is the difference in free energy between the products and the reactants.


ΔG = ΔH – TΔS

If ΔG is negative, free energy is released.

If ΔG is positive, free energy is required.

• If free energy is not available, the reaction does not occur.


Magnitude of ΔG depends on:

• ΔH—total energy added (ΔH > 0) or released (ΔH < 0).

• ΔS—change in entropy. Large changes in entropy make ΔG more negative.


If a chemical reaction increases entropy, the products will be more disordered.

Example: In hydrolysis of a protein into its component amino acids, ΔS is positive.


Second law of thermodynamics: disorder tends to increase because of energy transformations.

Living organisms must have a constant supply of energy to maintain order.


Exergonic reactions release free energy (–ΔG).

• Catabolism: complexity decreases (generates disorder).

Figure 8.3 Exergonic and Endergonic Reactions (Part 1)


Endergonic reactions consume free energy (+ΔG)

• Anabolism: complexity (order) increases.

Figure 8.3 Exergonic and Endergonic Reactions (Part 2)


In principle, chemical reactions can run in both directions.

• At chemical equilibrium, ΔG = 0

A B

The concentrations of A and B determine which direction will be favored.


Every reaction has a specific equilibrium point.

ΔG is related to the point of equilibrium: the further towards completion the point of equilibrium is, the more free energy is released.

ΔG values near zero are characteristic of readily reversible reactions.

Figure 8.4 Chemical Reactions Run to Equilibrium


ΔG also depends on the beginning concentrations of reactants and products, temperature, pressure, and pH.

ΔG is determined under standard conditions: 25°C, one atmosphere pressure, one molar (1M) solutions, and pH 7.


ATP (adenosine triphosphate) captures and transfers free energy.

ATP releases a large amount of energy when hydrolyzed.

ATP can phosphorylate, or donate phosphate groups, to other molecules.


Hydrolysis of ATP yields free energy:

ATP + H2O ADP + Pi + free energy

ΔG = –7.3 to –14 kcal/mol (exergonic)

Figure 8.5 ATP


Two characteristics of ATP account for the free energy released:

• Phosphate groups have a negative charge and repel each other—the energy needed to get them close enough to bond is stored in the P~O bond.

• The free energy of the P~O bond is much higher than the energy of the O—H bond that forms after hydrolysis.


Bioluminescence is an endergonic reaction driven by ATP hydrolysis:

Figure 8.5 ATP


The formation of ATP is endergonic:

ADP + Pi + free energy ATP + H2O

Formation and hydrolysis of ATP couples exergonic and endergonic reactions.


Coupling of exergonic and endergonic reactions is very common in metabolism.

Hydrolysis of ATP releases free energy to drive an endergonic reaction.

Figure 8.7 Coupling of ATP Hydrolysis to an Endergonic Reaction


An active cell needs to produce millions of molecules of ATP per second.

An ATP is typically consumed within a second of its formation.

Each ATP molecule undergoes about 10,000 cycles of synthesis and hydrolysis every day!


Catalysts speed up the rate of a reaction.

The catalyst is not altered by the reactions.

Most biological catalysts are enzymes (proteins) that act as a framework in which reactions can take place.


Some reactions are slow because of an energy barrier—the amount of energy required to start the reaction, called activation energy (Ea).

Activation energy puts the reactants in a reactive mode called the transition state.

Figure 8.8 Activation Energy Initiates Reactions


Activation energy changes the reactants into unstable forms with higher free energy—transition state intermediates.

Activation energy can come from heating the system—the reactants have more kinetic energy.

Enzymes and ribozymes lower the energy barrier by bringing the reactants together.


Enzymes and ribozymes are highly specific.

Reactants are called substrates.

Substrate molecules bind to the active site of the enzyme.

The three-dimensional shape of the enzyme determines the specificity.

Figure 8.9 Enzyme and Substrate


The enzyme–substrate complex (ES) is held together by hydrogen bonds, electrical attraction, or covalent bonds.

E + S ES E + P

The enzyme may change while bound to the substrate but returns to its original form.


The dissociation constant (KD) is a measure of the affinity of two molecules.

The lower the KD, the tighter the binding.

For enzymes and their substrates, KD values range from 10–5 to 10–6 M. This favors the formation of ES.


Enzymes lower the energy barrier for reactions.

The final equilibrium does not change, and ΔG does not change.

Enzymes can increase reaction rates by 1 million to 1017 times!

Figure 8.10 Enzymes Lower the Energy Barrier


In catalyzing a reaction, an enzyme may use one or more mechanisms:

• Orienting substrates

• Inducing strain in substrates

• Temporarily adding chemical groups

Figure 8.11 Life at the Active Site (Part 1)

Enzymes orient substrate molecules, bringing together the atoms that will bond.


Enzymes can stretch the bonds in substrate molecules, making them unstable.


Enzymes can temporarily add chemical groups to substrates.


Acid–base catalysis: enzyme side chains transfer H+ to or from the substrate, causing a covalent bond to break.

Covalent catalysis: a functional group in a side chain bonds covalently with the substrate.

Metal ion catalysis: metals on side chains loose or gain electrons.


Enzymes are much larger than their substrates and the active site is usually small.

The shape of the active site allows a specific substrate to fit precisely.


Substrates bind to active sites by hydrogen bonds, attraction and repulsion of electrically charged groups, and hydrophobic interactions.

Induced fit: enzyme changes shape when it binds the substrate, which alters the shape of the active site.

Figure 8.12 Some Enzymes Change Shape When Substrate Binds to Them


Some enzymes require “partners”:

• Prosthetic groups: non-amino acid groups bound to enzymes

• Inorganic cofactors: ions permanently bound to enzyme

• Coenzymes: small carbon-containing molecules; not permanently bound

Table 8.2


The rate of a catalyzed reaction depends on substrate concentration.

Concentration of an enzyme is usually much lower than concentration of a substrate.

At saturation, all enzyme is bound to substrate; it is working at maximum rate.

Figure 8.13 Catalyzed Reactions Reach a Maximum Rate


Maximum rate is used to calculate enzyme efficiency: Molecules of substrate converted to product per unit time (turnover number).

Turnover ranges from 1 to 40 million molecules per second!


The thousands of chemical reactions occurring in cells are organized in metabolic pathways. Each reaction is catalyzed by a specific enzyme.

The pathways are interconnected.

Regulation of enzymes and thus reaction rates helps maintain internal homeostasis.


Complicated metabolic pathways can be modeled using computer algorithms.

This new field is called systems biology.

Figure 8.14 Metabolic Pathways


Inhibitors regulate enzymes: Molecules that bind to the enzyme and slow reaction rates.

Naturally occurring inhibitors regulate metabolism.


Irreversible inhibition: inhibitor covalently bonds to side chains in the active site and permanently inactivates the enzyme.

Example: DIPF or nerve gas

Figure 8.15 Irreversible Inhibition


Reversible inhibition: inhibitor bonds noncovalently to the active site and prevents substrate from binding.

Competitive inhibitors compete with the natural substrate for binding sites.

Degree of inhibition depends on concentrations of substrate and inhibitor.

Figure 8.16 Reversible Inhibition (Part 1)


The cancer drug methotrexate is a competitive inhibitor.

It binds to the enzyme that catalyzes formation of a coenzyme for purine formation.

(Purines are needed for DNA replication and cell division.)

In-Text Art, Ch. 8, p. 158


Uncompetitive inhibitors bind to the enzyme–substrate complex, preventing release of products.

Noncompetitive inhibitors bind to enzyme at a different site (not the active site).

The enzyme changes shape and alters the active site.

Figure 8.16 Reversible Inhibition (Part 2)


Allostery: enzymes have different shapes.

Allosteric regulation: an effector binds enzyme at a site different from the active site, which changes its shape.

Active form—can bind substrate.

Inactive form—cannot bind substrate but can bind an inhibitor.

Figure 8.17 Allosteric Regulation of Enzymes


Most allosteric enzymes are proteins with quaternary structure.

The active site is on the catalytic subunit.

Inhibitors and activators bind to other polypeptides called regulatory subunits.


Some allosteric enzymes have multiple subunits with active sites. Substrate binding at one site can have allosteric effects, and reaction rate increases.


Reaction rates versus substrate concentration are very different than for nonallosteric enzymes.

In-Text Art, Ch. 8, p. 159 (1)

In-Text Art, Ch. 8, p. 159 (2)


For allosteric enzymes, reaction rate is very sensitive to substrate concentration (over a certain range).

They are very sensitive to low concentrations of inhibitors.

Thus they are important in regulating metabolic pathways.


Metabolic pathways:

• The first reaction is the commitment step—other reactions then happen in sequence.

• Feedback inhibition (end-product inhibition): the final product acts as a noncompetitive inhibitor of the first enzyme, which shuts down the pathway.

8 Working with Data: How Does an Herbicide Work?

The weed-killer glyphosate inhibits an enzyme (EPSP synthase) in the metabolic pathway used to synthesize several amino acids.

Experiment: Measure rate of the synthesis reaction in the presence of different concentrations of glyphosate and substrate (PEP).

Working with Data 8.1, Figure A

Working with Data 8.1: How Does an Herbicide Work?

Question 1:

At about what substrate concentration does EPSP synthase become saturated when no glyphosate is present?

How much substrate is needed to saturate EPSP synthase in the presence of 18 μM glyphosate?

In each case, what is the reaction rate at saturation?


Question 2:

Looking at the curve for the reaction rate without inhibitor, is EPSP synthase a multi-subunit allosteric enzyme?

Explain your answer.


Question 3:

Based on these data, what is the most likely mechanism for glyphosate inhibition of EPSP synthase: competitive, noncompetitive, or uncompetitive?

Why?

Figure 8.18 Feedback Inhibition of Metabolic Pathways


Many enzymes are regulated through reversible phosphorylation.

Enzymes can be activated when protein kinase adds a phosphate group and deactivated by protein phosphatase.


Every enzyme is most active at a particular pH.

pH influences the ionization of functional groups.

Example: at low pH (high H+) —COO– may react with H+ to form —COOH which is no longer charged; this affects folding and thus enzyme function.

Figure 8.19 pH Affects Enzyme Activity


Every enzyme has an optimal temperature.

At high temperatures, noncovalent bonds begin to break.

Enzymes can lose tertiary structure and become denatured.

Figure 8.20 Temperature Affects Enzyme Activity


Isozymes: enzymes that catalyze the same reaction but have different properties, such as optimal temperature.

Organisms can use isozymes to adjust to temperature changes.

Enzymes in humans have higher optimal temperature than enzymes in most bacteria—a fever can denature the bacterial enzymes.

8 Answer to Opening Question

Commercial use of purified enzymes is a multibillion-dollar industry.

Many come from bacteria and fungi—they are easy to grow in large quantities for industry, and the enzymes are easy to extract.

Documents

8 Energy, Enzymes, and Metabolism. 8 Energy, Enzymes, and Metabolism 8.1 What Physical Principles Underlie Biological Energy Transformations? 8.2 What