CHAPTER 3 Bioenergetics, Enzymes, and Metabolism.

CHAPTER 3

Bioenergetics, Enzymes, and Metabolism

3.1 Bioenergetics

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

Energy (thermodinamics)-- the ability/capacity to do work-- the ability to cause specific change

-- Type of EnergyKinetics:energy of movement

(Heat and light energy, 37C)Potential:energy of reorganization of atoms in molecules

(Chemical reaction energy)

02

1.Synthetic

Chemical bonds

Six kinds of workSix kinds of work

Biosynthesis


2.Mechanical

--Muscle

--Cell movement

--Chromosome movement

--Cytoplasmic streaming


3. Concentration

Passive Transport – no energy is expended and molecules move down the concentration gradient.DiffusionOsmosisHypotonicHypertonicIsotonic

Facilitated DiffusionActive Transport – energy (in the form of ATP) must be used to move certain molecules across the membrane.ExocytosisEndocytosisPhagocytosisPinocytosisReceptor mediated endocytosis


4. Electricalspecialized concentration work


5. HeatEspecially important to warm-blooded

animals

Six kinds of workSix kinds of work6. Bioluminescence

Especially important to produce ATPLighting bugs (fireflies) Bacterias

Dinoflagella ( marine algae)

Oxidation-------blue light color

luciferin

The Flow of Energy Through the Biosphere

Aerobic orAnaerobic

Oxidations and Reductions

Oxidations: removal of electrons from substances. (removal of Hydrogen and addition of Oxygen).

Reductions: addition of electrons from substances. (addition of Hydrogen and removal of Oxygen).

C6H1202

C02

CH4

OxidationReduction

C6H1202

CH4

Release of energy (heat)

Chemotrophs Phototrophs

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.


• 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.


• 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.

- Bioluminescence.


• 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.

Open and Closed Systems

Biological Systems==== Energy associated with Chemical Reactions ==== Work is the use of energy ( any type) to drive/

produce any process.Example: Oxidation of Glucose at the cellular level

1st Law of Thermodynamics Conservation of Energy (E)

ΔE = Eproducts – Ereactants (reactives)

ΔE = Change in total internal energy of a system

E: total internal energy of any given system

Reactants ProductsTotal Eproducts ; Total Ereactants (reactives)

1st Law of Thermodynamics Conservation of Energy

ΔH = ΔE + Δ(PV)

P = pressure, V = volumeΔH = change in heat content (enthalpy)

Reactants Products

E: H (Q) - PV(W) ; H(heat/enthalpy) = E + PV

1st Law of Thermodynamics Conservation of Energy

ΔH ~ ΔE

---Negative ΔH -- exothermic --heat is released

---Positive ΔH -- endothermic

Reactants ProductsChange on V and P is irrelevant

Heat

ΔH= Hprod - Hreact

---Negative ΔH -- exothermic --heat is released

---Positive ΔH -- endothermic

Example:

Gasoline + 02 (OXIDATION) CO2 + H2O

Heat

Photosynthesis

Heat (sunlight)

(lost)

(gain)


ΔH~ ΔE

Example 1:Cellulose (paper) + match +O2 burning (heat+glucose monomer +CO2)

ΔH~is simply a meausre of total enthalpy (heat) in any given system.

Example 2:Glucose-6-phospahte Fructose-6-phosphate

Thermodynamically favored (spontaneous?)

Can occur (possible) without input of additional energy

Directionality

Does not predict whether an energy change will be positive or negative.

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.

2nd Law of Thermodynamics

Gain in Disorder

Gain in disorder => entropy (S) ----CHAOS---

ΔS change in entropy between initial & final states

Example 3:

Cellulose (paper) + match +O2 combustion (heat+glucose monomer +CO2)

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

heat

ΔG = Free energy (G) = change during a process in energy available to do work

ΔH = ΔG + TΔS

ΔH = change in enthalpyΔS = change in entropy between initial & final statesT = temperature in Kelvins (K= oC +273)

Combining the 1st and 2nd Laws

ΔG = ΔH - TΔS

spontaneity

Combining the 1st and 2nd Laws

ΔG = ΔH - TΔS

Negative ΔG = exergonic(energy production) => spontaneousCAN GO-possible (WILL GO)

Positive ΔG = endergonic (energy requiring) => not spontaneousCAN NOT GO-not possible (WILL NOT GO)

0 ?

+

-

G

Figure 5-9 Dependence of ∆G on the Signs and Numerical

Values of ∆H and the Terms (T∆S)

Negative ΔH -- exothermic (heat)Positive ΔH -- endothermic

Figure 5-10 Changes in Free Energy for the Oxidation and Synthesis of Glucose

Chemotrophs Prototrophs

+ Energy + Energy

Free-Energy Changes in Chemical Reactions (1)

• All chemical reactions are theoretically reversible.

• All chemical reactions spontaneously proceed toward equilibrium (Keq = [P]/[R]).

• 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).

R P

At equilibrium, ΔG = 0

[A][B] = [C][D]

k1 = k2

A + B C + Dk1

k2

Free Energy Changes in Chemical Reactions

K1 rate constant of reactantsK2 rate constant of products

AB reactantsCD products

At equilibrium

k1[A][B] = k2[C][D]

k1 = [C][D]

k2 = [A][B] Keq =k1

k2

At equilibrium, free energy to do work – minimumentropy - maximum

Calculating G

Free Energy Changes in Chemical Reactions at standard conditions

T = 298°KP = 1 atmosphereReactants & products = 1MWater = 55.6MpH = 7

ΔG°′ = standard free energy change

?

Free Energy Changes in Chemical Reactions at standard conditions

ΔG°′ = -RT lnK′eq

R = gas constant (1.987 cal/mol-K)T = absolute temperature

ΔG°′ = -2.303RT logK′eq

if equilibrium constant > 1, negative ΔG°′

if equilibrium constant < 1, positive ΔG°′

ln x= 2.303 log x

The Relationship Between ∆G°´ and K´eq

[P] [R]Keq =

R P

Calculating G’

Glu-6-P Fruct-6-P

Keq=0.5

G’ =-954 cal/mol

Products

Reactants

Non equilibrium

Our exp. conditions

G’: Go’ + R T ln [C][D]

[A][B]

R(A and B) P(C and D)

Free Energy Changes in Chemical Reactions in the cell

ΔG′ = ΔG°′ + 2.303RT log[C][D]

[A][B]

Example: ATP hydrolysis: ATP ADP + Pi ΔG°′ = -7.3 kcal/mol

[ATP] = 10 mM, [ADP] = 1 mM, [Pi] = 10 mM

ΔG’ = ΔG°′ + 2.303RT log[ADP][Pi]

[ATP]

ΔG’ = -7.3 kcal/mol + (1.4 kcal/mol) log[10-3][10-2]

[10-2]ΔG’ = -11.5 kcal/mol

The Meaning of ∆G°´ and ∆G´

Coupling Endergonic & Exergonic Reactions

Large Positive ΔG′ energy input

Example:Glutamine from Glutamic acid

Glutamic acid + NH3 GlutamineΔG′ = +3.4 kcal/mol

2 sequential reactions:Glutamic acid + ATP glutamyl phosphate + ADPGlutamyl phosphate + NH3 glutamine + Pi

Glutamic acid + ATP + NH3 glutamine + ADP + Pi

ΔG′ = -3.9 kcal/mol


Large Positive ΔG′ energy input

Example:Glutamine from Glutamic acid (glutamine synthase)

Glutamic acid + NH3 GlutamineΔG′ = +3.4 kcal/mol

2 sequential reactions:Glutamic acid + ATP glutamyl phosphate + ADPGlutamyl phosphate + NH3 glutamine + Pi

Glutamic acid + ATP + NH3 glutamine + ADP + Pi

ΔG′ = -3.9 kcal/mol

Common intermediate

Product of the first reaction

becomes

Substrate for the second


Free-Energy Changes in Chemical Reactions (2)

• Free energy changes of reactions are compared under standard 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. – 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.

Coupling Endergonic and Exergonic Reactions

Coupling Endergonic and Exergonic Reactions

ATP hydrolysis

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.

Equilibrium vs. Steady State

ΔG°′= At equilibrium, free energy to do work – minimum entropy – maximum

ΔG′ = Steady state – concentrations of reactants and products remain relatively constant, even though individual reactions are not necessarily at equilibrium

Reactions generally don’t reach equilibrium in the cell

Steady State versus Equilibrium

ΔG°’= + 410 cal/mol

Glucose-6-phosphate ? Fructose-6-phosphate

25ºC at a concentration of 1 M of both reagentswith a Keq=0.5 IN VITRO

37ºC at a concentration of 83 uM and 14 uM of reagentswith a Keq=0.169 IN VIVO=CELL

Glucose-6-phosphate Fructose-6-phosphate

Enzymes

ΔG’= - 644 cal/mol

Negative ΔG = exergonic => spontaneousPositive ΔG = endergonic => not spontaneous

CAN GO (WILL GO)

Reactants Products(more energy) (less energy)

Heat

Negative ΔH ---- exothermic --heat is released

Example: Glucose oxidation and ATP hydrolysis

G=-686 kcal/mol -7.3kcal/mol

Enzymes

3.2 Enzymes as Biological Catalysts

• Enzymes are catalysts that speed up chemical reactions.

• Enzymes are NOT always proteins.

• Enzymes may be conjugated with

non-protein components.– Cofactors are inorganic enzyme conjugates.– Coenzymes are organic enzyme conjugates.

RibozymesRibozymes (RNA/protein)(RNA/protein)

Properties of Enzymes (1)• 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. Only changes rate at which equilibrium is achieved. Can’t drive endergonic reaction.

• Are highly specific for their particular reactants called substrates. Forms transient complexes with substrate.

• Produce only appropriate metabolic products.• Can be regulated to meet the needs of a cell. Enzymatic activity of EGFR

• Lowers EA allowing reaction to occur without thermal activation.

Properties of Enzymes (2)

*

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. ATP+H2O ----- -----ADP+Pi

[ATP:H2O]

Metastable (intermediate chemical) state – condition where reactants are thermodynamically unstable but have insufficient energy to exceed the activation energy barrier for the reaction

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. ATP+H2O ----- ---ADP+Pi

[ATP:H2O]

Catalyst – agent that enhances the rate of a reaction by lowering the energy of activation

Enzyme=Catalyst (WILL GO)

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.

Kinetic energy is proportional to theTemperature under our exp. conditions

25C

NON-catalyzed reaction

EA

Catalyzed reaction

EA

37C

The Active Site

• 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.

The Active Site (~Motif)

307 amino acidActive siteHis, Glu, Arg, Tyr

130 amino acidActive siteGlu, Asp

Enzyme structure

May have prosthetic groups (small organic molecules or metal ions)

(often electron acceptors)

active (Catalytic) site

Substrate specificity

Temperature dependent

pH sensitivity

The Major Classes of Enzymes with an Example of Each

Mechanisms of Enzyme Catalysis (1)

• Substrate orientation means enzymes hold substrates in the optimal position of the reaction.


• 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.

Two Models for Enzyme-Substrate Interaction


• 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.

The Catalytic Cycle of an Enzyme

Enzyme KineticsS P enzymes

Ef + S EbS Ef + Pk1 k3

k2

--initial reactions rates (very early in the chemical reactions)

[S]>>>>>>>>>>>>>>>[P]

Enzyme Kinetics (1)

• 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 is 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.

Enzyme Kinetics (2)

• The Michaelis constant (KM) is the substrate concentration at one-half of Vmax.– Units of KM are

concentration units.– The KM may reflect

the affinity of the enzyme for the substrate.

Experimental Procedure for Studying the Kinetics of the Hexokinase Reaction

Glu + ATP (1mM) Glu-6-phosphate +ADPEnz=1uM

The Relationship Between Reaction Velocity and Substrate Concentration

--Low substrate concentration [S]<<Km

v is proportional to [S]

--High substrate concentration [S]>>Km

v independent of [S]

--[S] = Km: [S] where v = ½ Vmax

Michaelis-Menten equation

Vmax: maximum velocity of reaction[S]: initial substrate concentrationv: initial velocityKm: Michaelis constant

substrate concentration @ ½Vmax

Vmax is an approximation number (~Vmax)

The Relationship Between Reaction Velocity and Substrate Concentration

Turnover number (K catalysis): Kcat

Kcat = (cat=catalysis) rate at which substrate molecules are converted to product by a single enzyme molecule operating at Vmax

units: time (sec)-1

Kcat = Vmax/[Et]

[Et]. Kcat=Vmax

[Et] = enzyme concentration (Molar [M])

Km and kcat Values for Some Enzymes

KM = Kcat

Double reciprocal plot

v = (Vmax[S])/(Km + [S])

1/v = (Km + [S])/(Vmax[S])

1/v = Km 1 + [S] Vmax [S] Vmax[S]

1/v = Km 1 + 1 Vmax [S] Vmax

Vmax is an approximation number (~Vmax)

Enzyme Kinetics (3)

• 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 Kinetics (4)

1/v = Km 1 + 1 Vmax [S] Vmax

Experimental Procedure for Studying the Kinetics of the Hexokinase Reaction

Glu + ATP Glu-6-phosphate +ADP

Double-Reciprocal Plot for the Hexokinase

Vmax = 10 µmol/min

-1/Km = -6.7

Km = 0.15mM

The Effect of Temperature and pH on the Reaction Rate of Enzyme-Catalyzed Reactions

Enzyme Kinetics (4)

Enzyme Inhibitors (1)

• 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.

Modes of Action of Competitive and Noncompetitive Inhibitors

Reversible – noncovalentE + I EI


• 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.

Ethanol in Alcoholic beverages (BEER)

Alcohol Dehydrogenase(ADH)

Aldehyde Dehydrogenase

Low Km ---mitochondrial

Low/High Km-- cytosolic/ stomach/ liver

--OH --C=O C00-

ALDH

ALDH

ALDH

Hangover

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.

(Penicillin and derivatives=irreversible Inhibitor)– Components of the system by which bacteria duplicate,

transcribe, and translate their genetic information (streptomycin=bacterial ribosomes, ciprofloxacin=bacterial DNA synthesis)

– Enzymes that catalyze metabolic reactions specific to bacteria (Sulfa drugs and folic acid); Protease and reverse transcriptase inhibitors (HIV)

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.

3.3 Metabolism

• Metabolism is the collection of bio-chemical reactions that occur within a cell via enzymes.

• 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.

An Overview of Metabolism (1)

• 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.

An Overview of Metabolism (2)

• 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)

---SH HS------ ---S-S---

Metabolic Regulation (1)

• 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 (inhibited or stimulated) by compounds binding to allosteric sites # active site.

• In feedback inhibition (negative feedback), the product of the pathway allosterically inhibits one of the first enzymes of the pathway.

Metabolic Regulation (2)

Allosteric effector – small organic molecule that regulates the activity of an enzyme for which it is neither the substrate nor the intermediate product

(Rate limiting enzymes)

Mechanisms of Allosteric Inhibition and Activation

Inhibition Activation

Mechanisms of Allosteric Inhibition and Activation

Catalytic & Regulatory Subunits

Enzyme Regulation

Allosteric regulation

Covalent modification

Enzyme Regulation – Covalent modification

Phosphorylation

Methylation

Acetylation

Proteolytic cleavage

The Regulation of Glycogen Phosphorylase by Phosphorylation

Activation of Pancreatic Zymogens by Proteolytic Cleavage

Autoactivation

Pancreatitis

Basic Concepts on Enzymatic Action

1-Very Active-Powerful

2-Highly Specific-Catalysis

CO2 + H2O CO3H2

1 molecule per sec

Plus enzyme (carbonic anhydrase, CA)

7.7 x 10 6 sec

Time

[P]

A P

Enz

No Enz

Sec Hours

3-Examples

A- transport of CO2 in blood/tissue

CO2 + H2O CO3H2 CO3H- + H+


Tissues

Lung

C A in red cells

C A in red cells

3-Examples

B- Sodas


--bubbles in soda

--bubbles in mouth after drinking

Time

[P]

A PEnz

[A]

Time

[P]

[Enz]

[P] is proportional to [A] and [Enz]

RNA splicing:removal of introns from pre-RNA

EnzymesEnzymes(Proteins)(Proteins)

RibozymesRibozymes(RNA)(RNA)

(1981)(1981)

CHAPTER 11Pages 449-454

Spliceosomes remove introns from pre-mRNA.Spliceosomes is a particle formed by RNA (snRNA) and Proteins (sm Proteins)

15% diseases(Lupus Erythematosus)

Transcription by RNAPolymerase II and capping

E

I

mG

mG

mG

mGAAA

AAA

AAA

Removal of 3’ end by nuclease and polymerase

Ligation of exons

EndonucleolyticCleavage at splice junctions

Processingintermediate

ISplice site: 5’ Splice site: 3’

EE...G GU...........................A..........................AG G

Branch site sequence

95-99%

5-1%?

...G GU...........................A..........................AG G

hydroxyl, 2’Phosphoryl 5’

Transesterification ........... Required limited amount of ENERGY

Eukaryotes: Prokaryotes: + _

U3?rRNA

CHAPTER 3 Bioenergetics, Enzymes, and Metabolism.

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Transcript of CHAPTER 3 Bioenergetics, Enzymes, and Metabolism.