Chapter 8

Introduction to Metabolism

Metabolism is the set of life-sustaining chemical transformations within the cells of living organisms. These enzyme-catalyzed reactions allow organisms to grow and reproduce, maintain their structures, and respond to their environments.

1a. Using graphs and words, compare and contrast Exergonic vs. Endergonic chemical

reactions

Exergonic reactions are those that release energy. During these reaction the reactant has higher free energy than the product. This is expressed by -G (negative Gibbs number). These reaction occurs spontaneously and their rate is usually increased by enzymes. An example is the breakdown of ATP into inorganic phosphate ion and ADP during exerting work. ATP----> ADP + Pi+ energy

Endergonic reactions are those that store energy. During these reactions the reactant has lower free energy than the product. This is expressed by +G (positive Gibbs number). They do not happen spontaneously and need supply of energy to occur. Example would be production of ATP during aerobic respiration: ADP+ Pi + energy -----> ATP

Because one reaction releases and one requires energy, many times reactions that are endergonic and exergonic are coupled together (such as redox reactions)

An exothermic reaction can be either endergonic or exergonic. The same is also true for an endothermic reaction, as in your example. You'd have delta H > 0 (endothermic) but delta G < 0 (exergonic). 

Exergonic vs. Endergonic

• More energy in reactants than products.

• Delta G negative• Energy is released• Catabolic• Spontaneous• Increased stability

(increased entropy)

• More energy in products than reactants.

• Delta G positive• Energy is added• Anabolic• Non-spontaneous• Decreased stability

(decreased entropy)

1b. Chemical reactions with and without an enzyme

With an enzyme:

• Energy of activation is lowered

• Speed of reaction is faster

• Energy in products and reactants are unchanged

• The sign of delta G does not change.

• A non-spontaneous reaction does not become spontaneous.

2. Explain how cells are able to remain alive and increase in complexity in accordance with the

second law of thermodynamics• 2nd law: Disorder (entropy) in the universe is continuously

increasing.• In general, energy transformations proceed

spontaneously to convert matter from a more ordered, less stable form, to a less ordered, more stable form.

• To remain alive, delta G cannot be equal to 0. This is done by coupling endergonic chemical reactions that increase or maintain complexity to exergonic chemical reactions that release energy. Product becomes reactant for the next chemical reaction

• . Heterotrophic organisms breakdown organic compounds (chemical bonds within compounds are sources of potential energy). Autotrophic organisms convert one form of energy (photons of light or energy in inorganic molecules) to another form: chemical bonds in organic molecules (again, a source of potential energy-stored).

3. Compare the strategies employed by different lineages of cells to acquire and

utilize free energy• Autotrophs: Photoautotrophs that convert sunlight into

chemical energy (acquire); chemoautotrophs that convert inorganic compounds into organic molecules (acquire). Stored in new chemical bonds. Released to do work.

• Heterotrophs: Breakdown ingested organic compounds (acquire)via aerobic cell respiration. Herbivores vs. carnivores vs. decomposers

• Heterotrophs: Breakdown ingested or absorbed (acquired) organic compounds via anaerobic cell respiration/fermentation.

• Utilize this acquired free energy to power cellular activities: transport/movement /chemical reactions.

4. How does ATP perform work?

Composition of ATP

a) 5 carbon sugar – ribose

b) adenine (purine)

c) triphosphate group: PO4- x 3

ATP

• A) negative charges of phosphate groups repulse one another in a tight area. This causes

• B) 2 covalent bonds between phosphate groups to be unstable. Coiled spring analogy: phosphates straining away from one another.

• C) These 2 bonds have low activation energy and are easily broken through hydrolysis.

• Only outermost high energy phosphate bond is hydrolyzed, cleaving off phosphate group at the end.

• ATP + water = ADP (adenosine diphosphate) + inorganic phosphate

• In a test tube, this reaction generates a change in free energy of – 7.3 kcal/mole

• In a cellular environment, delta G = -13 kcal/mole

ATP hydrolysis: coupling by phosphate transfer E.G. Glutamic Acid + Ammonia to Glutamine

• ATP hydrolysis is coupled to an endergonic process (energy requiring) by transferring a phosphate group from ATP to some other molecule.

• Phosphorylation produces a phosphorylated intermediate.

• This intermediate is less stable than original molecule. This is energy coupling by phosphate transfer.

5. Using graphs and words, explain the effects of temperature and pH on enzyme activity

a) Temperature: Up to a point, increased temperature increases the rate of a chemical reaction by increasing the number of collisions between enzyme and substrate.

At some point, increased temperature stops chemical reaction by denaturing protein by disrupting ionic, H bonds, weak interactions.

Each enzyme has an optimum range of temperatures in which to function.

pH

• Each enzyme has an optimum pH range usually between 6 – 8. Exception: pepsin which begins the digestion of protein in the mammalian stomach. Optimal pH is 2.0

• A pH that is out of the range may denature an enzyme, causing it to lose it’s secondary and tertiary structures and resulting in the loss of activity.

6. Using a graph and words, how does increased substrate concentration affect enzyme activity?

Rate of Conversion of Substrate

• Partly dependent on initial concentration of substrate. Increased concentration, increases the reaction rate because of the increased chance of contact.

• Substrate concentration can affect the rate of reaction until all of the active sites are filled (saturated).

Rate of Saturation

Reaction

Substrate Concentration

Examples of Competitive and non-competitive inhibition

• An inhibiting molecule structurally similar to the substrate molecule binds to the active site, preventing substrate binding. Eg. Inhibition of folic acid synthesis in bacteria by the sulfonamide (antibiotic) Prontosil. E.g. Carbon monoxide binds to the active site of hemoglobin and is a competitive inhibitor that binds irreversibly.

• An inhibitor molecule binding to an enzyme (not to its active site) that causes a conformational change in its active site resulting in a decrease in activity. Inhibitor does not have to be chemically similar to substrate.

• Eg. Metal ions disrupting disulfide bridges in many enzymes including cytochrome oxidase (enzyme in electron transport chain). Hg2+, Ag+, Cu2+ bind to –SH groups, breaking –S-S- linkages; changes shape of the active site.

7. Competitive vs. Non-competitive inhibitors

• Competitive: chemically similar; competes with substrate for active site. Effect can be diluted with addition of substrate.

• Non-competitive: does not have to be chemically similar. Binds somewhere else on enzyme that is not the active site. Changes the active site so that it does not bind substrate in the way that it did without an inhibitor.

8. Explain allosteric regulation of enzymes as an example of feedback inhibition (negative feedback)

• A particular type of non-competitive inhibition of biochemical pathways

• End-product binds to first enzyme in pathway and shuts the pathway down.

• Also called: End-product inhibition; a version of negative feedback

• Mechanism of homeostasis

• The allosteric site is a specific portion of an enzyme. Not the active site.

• Serves as an on-off switch. Binding of a substance to this site can switch an enzyme between active and inactive configuration.

2 Examples of allosteric enzymes: phosphofructokinase and threonine deaminase

• Pathway is switched off by its end-product which acts as an inhibitor.

• This is a form of non-competitive inhibition.

• Shape of the allosteric enzyme can be altered by the binding of end products to an allosteric site, decreasing its activity.

• E.g. ATP inhibition of phosphofructokinase in glycolysis where ATP turns off its own production.

Other things to worry about:

• Models of enzyme specificity

• Equation for free energy

• Types of work that cells do

• Examples of how active sites are involved in catalysis.

• Use of enzymes in biotechnology

• E.g. Lactase, pectinase, proteases

1. METABOLISM

Definition: all of the 1000s of precisely coordinated, complex, efficient and integrated chemical reactions in an organism.

Metabolic pathways: chemical reactions ordered into sequenced branching routes controlled by enzymes.

a) Metabolic pathways that release energy by breaking down complex molecules to simpler molecules: CATABOLIC PATHWAYS.

E.G. Cellular respiration where glucose and other organic fuels broken down to carbon dioxide and water; energy is released to do work.

Metabolic pathways continued

b) Metabolic pathways which consume energy to build complex molecules from simpler ones: Anabolic Pathways.

e.g. Synthesis of proteins from amino acids (for defense, motion, transport).

Common to Both Pathways: the Involvement of Energy

• Energy coupling: energy released from catabolic pathways fuel anabolic pathways.

2. Bioenergetics: analysis of how energy powers the activities of

living organismsEnergy: the capacity to do workEnergy exists in 2 states: a) Kinetic energy: energy of motion.

Moving objects perform work by causing other matter to move.

e.g. Flow of electrons: electricity; nerve which transmits signals; light- photosynthesis

KINETIC ENERGY

b) Potential energy: stored energy. Not moving but having the

capacity to move• The energy of position

c. Examples of energy forms

• Mechanical: mechanoreceptors = pressure, touch

• Heat: thermal receptors

• Sound: also a mechanoreceptor

• Electrical current

• Light: photoreceptors (eye)

• Radioactive radiation

• Chemical: chemoreceptors – taste, smell

d) All forms of energy can be converted to heat

• We can express energy in terms of heat.

• The study of energy: thermodynamics (heat change).

• Unit of heat most commonly used in biology: kilocalorie (1000 calories).

• Unit of heat in Physics is the joule = 0.239cal.

e) Energy flow into the biological world: the sun

• In photosynthesis: small molecules combine into more complex molecules. Energy is stored as potential energy in the covalent bonds between atoms in the sugar molecules.

f) During a chemical reaction, the energy stored in chemical bonds

may transfer to new bondsElectrons may pass from one atom or molecule

to another.Loss of electrons: oxidizedGain of electrons: reducedThese reactions always take place together:

oxidation-reduction reactions (redox).Redox reactions play a key role in the flow of

energy through biological systems because the electrons that pass from one atom to another carry energy with them.

• If electron is boosted into another energy level (light) the electron’s extra energy is transferred with it. Added energy is stored as potential energy that the atom can later release when the electron returns to its original energy level.

3. The Laws of Thermodynamics

A set of universal laws that govern all energy changes in the universe, from nuclear reactions to the buzzing of a bee.

a) The 1st law of thermodynamics: concerns the amount of energy in the universe. Energy can change from one form to another, but can never be destroyed nor created.

1st Law

• Potential energy can be converted to kinetic energy.

• During energy conversion, some energy is lost as heat.

• Energy flow in the biological world is unidirectional:

• Sun energy enters system and replaces energy lost from heat

b) 2nd Law of Thermodynamics

• Concerns the transformation of potential energy to heat, or random molecular motion

• Disorder (entropy) in the universe is continuously increasing.

• In general, energy transformations proceed spontaneously to convert matter from a more ordered, less stable form, to a less ordered, more stable form.

c. Entropy: measure of the disorder of a system

d) Free energy: the amount of energy actually available to break and form other chemical bonds…energy available to do work.

G = (free energy) after Gibbs

H = energy contained in a molecule’s chemical bonds (enthalpy)

S = Entropy (the energy unavailable because of disorder)

T = Temperature in degrees Kelvin

Free Energy Equation

• G = H – TS

• Change in free energy (delta G): Change in H – T x change in S.

• Change in free energy positive number: products of reaction has more energy than reactants. Endergonic chemical reaction (energy requiring) because either H has increased and/or S has decreased.

Change in Free energy: Negative

• Change in G negative number: products of reaction have less energy than reactants. Exergonic (energy liberating) chemical reaction. Spontaneous

Free Energy characteristics

Exergonic vs. Endergonic

e) Activation Energy

• Required energy input to begin a chemical reaction, even if chemical reaction is spontaneous.

• Catalysis: process of influencing chemical bonds in a way that lowers the activation energy needed to initiate a reaction.

• Catalysts speed up the process of chemical reactions without being changed by the reaction.

With and Without Enzyme

4. Catalysts• A) do not violate the laws of thermodynamics.

Endergonic reaction will not occur spontaneously.

• B) decrease activation energy• C) accelerate both forward and reverse

reactions by exactly the same amount.• D) do not determine the direction of the chemical

reaction which is determined solely by the difference in free energy. Does not alter proportions of reactants converted to products.

• E) Do not change which chemical reactions are spontaneous.

Summary: Exergonic vs. Endergonic Reactions

1. Net release of free energy

2. Delta G = -number

3. Can be spontaneous

4. E.g. cell respiration

5. E.g. 1 mole of glucose contains

Delta G = -686 Kcal/mol

1. Absorbs free energy from surroundings

2. Delta G = + number

3. Nonspontaneous

4. E.g. photosynthesis

5. Eg. CO2 + H20

glucose

Delta G = +686Kcal/mol

More Endergonic vs. Exergonic

If a reversible process is endergonic in one direction, reverse process is exergonic

At equilibrium, the change in free energy = 0As a result you can do no work; dead!In living cells, delta G is never allowed to reach 0Product of 1 reaction becomes the reactant in

another reaction on down the pathway.Cell Respiration is driven by: great difference

between the free energy of glucose and the free energy of carbon dioxide and water.

Hydroelectric Analogy

5. ATP

• Chief energy currency that all cells use; most of the energy harvested in plants is used to manufacture ATP.

• ATP = Adenosine triphosphate

• A)Composition of ATP

a) 5 carbon sugar – ribose

b) adenine (purine)

c) triphosphate group: PO4- x 3

ATP Structure

More on the triphosphate group

• A) negative charges of phosphate groups repulse one another in a tight area. This causes

• B) 2 covalent bonds between phosphate groups to be unstable. Coiled spring analogy: phosphates straining away from one another.

• C) These 2 bonds have low activation energy and are easily broken through hydrolysis.

b) Reactions involving ATP:

• Only outermost high energy phosphate bond is hydrolyzed, cleaving off phosphate group at the end.

• ATP + water ADP (adenosine diphosphate) + inorganic phosphate

• In a test tube, this reaction generates a change in free energy of – 7.3 kcal/mole

• In a cellular environment, delta G = -13 kcal/mole

c) ATP hydrolysis is coupled

• ATP hydrolysis is coupled to an endergonic process (energy requiring) by transferring a phosphate group from ATP to some other molecule.

• Phosphorylation produces a phosphorylated intermediate.

• This intermediate is less stable than original molecule. This is energy coupling by phosphate transfer.

ATP Hydrolysis: Unstable Phosphorylated Intermediate

Example: Glutamic Acid to Glutamine

• The addition of ammonia to glutamic acid makes a different amino acid = glutamine

• This process is an endergonic process and requires energy.

• Delta G = +3.4 Kcal/mole• It is coupled to the phosphorylation of glutamic

acid by ATP, which transfers chemical instability to the amino acid.

• 2nd step: ammonia displaces the phosphate group from the intermediate, forming glutamine.

• ATP is regenerated

Regeneration of ATP

• Involves the conversion of ADP and inorganic phosphate to ATP and water

• Energy requiring process that is coupled to an exergonic pathway: cellular respiration.

• Let’s review an energy profile of a reaction

ATP Cycle

Exergonic Chemical Reaction

Enzyme Activity: A Closer Look

a) Enzymes are substrate specific

b) Definition of substrate: reactant upon which an enzyme acts

c) Enzymes convert reactants to products by joining with reactants or substrates

d) Enzymes can distinguish its substrate from closely related compounds such as isomers.

e) The part of the enzyme that binds to substrate is called the active site

The Active Site

a) May be only a few amino acids long

b) May be a pocket or groove on the surface of a protein

c) Enzyme specificity is based on its shape.

Models of Enzyme-Substrate Specificity

Lock and Key Fit between the shape and

chemistry of its active site and the shape of the substrate described as lock (enzyme) and key (substrate).

Implies rigidity. Shape is not flexible.

Each enzyme only binds to one substrate.

Induced Fit: more like a handshake. Active site is rigid; as substrate enters the active site, it is induced to change shape by the substrate. Result: active site fits even more snugly around the substrate. An enzyme might bind >1 substrate. Accounts for the broad specificity of some enzymes.

ENZYME INDUCED FIT

Advantage of induced fit model

• Induced fit brings chemical groups of the active site into positions that enhance their ability to work on the substrate and catalyze the chemical reactions.

The Active Site: Possible Modes of Action

1. Holds substrate using hydrogen and ionic bonds; weak interactions.

2. R groups of a few amino acids in active site catalyze conversion of substrate to product. Remember that enzyme is not changed by the reaction.

3. Able to convert more than a thousand molecules per second, some enzymes are faster.

4. In some cases, active site provides a template for the substrates to come together. It may align the substrate so that the substrate can interact with the template.

5. Active site might be a pocket of low or high pH

CATALYTIC CYCLE: Sucrase

Environmental Factors that Affect Enzyme Activity

a) Temperature: Up to a point, increased temperature increases the rate of a chemical reaction by increasing the number of collisions between enzyme and substrate.

At some point, increased temperature stops chemical reaction by denaturing protein by disrupting ionic, H bonds, weak interactions.

Each enzyme has an optimum range of temperatures in which to function.

ENZYME REGULATION

b) pH

• Each enzyme has an optimum pH range usually between 6 – 8. Exception: pepsin which begins the digestion of protein in the mammalian stomach. Optimal pH is 2.0

Speed of a chemical reaction

• Determined by the speed at which the active site can convert substrate to product or can manufacture more enzyme.

c) Inhibitors

• Chemicals other than intended reactant bonded to the active site or changing the shape of the active site.

• Two general types: Competitive and Noncompetitive

Competitive and Noncompetitive Inhibitors

Competitive Inhibitors

• An inhibiting molecule structurally similar to the substrate molecule binds to the active site, preventing substrate binding. Eg. Inhibition of folic acid synthesis in bacteria by the sulfonamide (antibiotic) Prontosil. E.g. Carbon monoxide binds to the active site of hemoglobin and is a competitive inhibitor that binds irreversibly.

Non-Competitive Inhibition

• An inhibitor molecule binding to an enzyme (not to its active site) that causes a conformational change in its active site resulting in a decrease in activity. Inhibitor does not have to be chemically similar to substrate.

• Eg. Metal ions disrupting disulfide bridges in many enzymes including cytochrome oxidase (enzyme in electron transport chain). Hg2+, Ag+, Cu2+ bind to –SH groups, breaking –S-S- linkages; changes shape of the active site.

Allosteric Inhibition

• A particular type of non-competitive inhibition of biochemical pathways

• End-product binds to first enzyme in pathway and shuts the pathway down.

• Also called: End-product inhibition; a version of negative feedback

• Mechanism of homeostasis

Allostery and control of metabolic pathways by end-product inhibition

• The allosteric site is a specific portion of an enzyme. Not the active site.

• Serves as an on-off switch. Binding of a substance to this site can switch an enzyme between active and inactive configuration.

• If a substance decreases its protein’s activity: allosteric inhibitor

• If a substance increases its protein’s activity: allosteric activator

• When an active site is stabilized by a substrate molecule: exhibits cooperativity.

ALLOSTERIC REGULATION

COOPERATIVITY

Feedback Inhibition

• Pathway is switched off by its end-product which acts as an inhibitor.

• This is a form of non-competitive inhibition. • Shape of the allosteric enzyme can be

altered by the binding of end products to an allosteric site, decreasing its activity.

• E.g. ATP inhibition of phosphofructokinase in glycolysis where ATP turns off its own production.

E.g. Pathway that converts threonine to isoleucine

• This pathway shuts down when isoleucine accumulates.

• Isoleucine is an allosteric inhibitor of the enzyme that catalyzes the first step in the pathway.

END PRODUCT INHIBITION

Location of Enzymes

• Some enzymes have defined locations (e.g. catalase – in microbodies/peroxisomes of all cells).

• Many enzymes located in membranes, such as the ones on the inner mitochondrial membrane (cellular respiration) and the chloroplast membranes (photosynthesis). Digestive enzymes embedded in the membranes of cells of the small intestine (brush border).

Use of enzymes in biotechnology: Example 1Lactase + Lactose

Intolerance• 10% of Americans

• 10% of Africa’s Tutsi tribe

• 50% of Spanish and French people

• 99% of Chinese people

Lactose Intolerance

• Lactase gene is switched off after weaning.• Stone-age ancestors of European dairy-lovers

couldn’t digest milk either. Looked at 7,000 year old fossils of ancient

Europeans and could not find in their DNA the lactose tolerance mutation. Seemed to have evolved lactose tolerance around 5,000 years ago.

Mutation arose independently in Africa around 7,000 years ago…very high frequency in Tanzanian Hadza population

Lactase

• Lactose is disaccharide found in milk.

• Converted by enzyme lactase to glucose and galactose.

• Lactase can be obtained from the yeast, Kluveromyces lactis.

• Used to help people who are lactose intolerant.

• Used to break down lactose in milk shakes and fruit yoghurt so that less sugar needs to be used (glucose and galactose are sweeter than lactose).

• Used in ice cream to create smoother texture (lactose crystallizes)

• Can be used in making of fermented cottage cheese and yoghurt by bacteria…faster.

Lactase can be used in 2 ways during food processing.

• 1. Added to milk. Final product contains the enzyme.

• 2. Immobilized on a surface or in beads of a porous material. Milk is allowed to flow past immobilized lactase. Avoids contamination of the product with lactase.

Examples of the use of proteins in biotechnology: The use of organisms or parts of organisms to produce things or to

carry out useful processes. • Pectinase: obtained from

fungus, Aspergillus niger • Breaks down the complex

polysaccharide, pectin, found in the cell walls of plants.

• Used during the crushing of fruit to make juice more fluid and easy to separate. (Prevents pectin from forming cross-links and trapping juice)

• Increases juice volume and less cloudy.

• Protease: obtained from a bacteria, Bacillus licheniformis, that is adapted to grow in alkaline conditions.

• Breaks down proteins into soluble peptides and amino acids.

• Used in detergents in laundry washing powders to digest protein.

• High pH optimum allows it to remain active in alkalis.

• Allows lower temps to be used, lower energy use, less shrinkage or loss of colored dyes.

Pectin

Review Guide Enzymes