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DEPARTMENT OF BIOTECHNOLOGY ENGINEERING

KULLIYYAH OF ENGINEERING

Course Title: Biochemistry II

Course Code: BTE 3430

Credit Hours: 3

Contact Hours: 3 Lecture (compulsory)

Instructor: ASSOCIATE PROFESSOR DR. FARIDAH YUSOF

Time: 5.00 to 6.20 pm Mondays and Wednesdays for 7 weeks, 1 week of break and 7

weeks (14 weeks)

Required Reading: Voet, D. and Voet, J. G. (2004)Biochemistry (Third Edition), John

Wiley and Sons, Inc.

Method of Evaluation: Mid-Term Exam 30%

Quizzes 10%

Assignments 10%

Final Exam 50%

TOTAL 100%

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METABOLISM

INTRODUCTION

Metabolism is the overall process through which living systems acquire and use free

energy to carry out their various functions Metabolism is divided into 2 parts - catabolism and anabolism

1. Catabolism

or degradation, in which nutrients and cell constituents are broken down to salvage

their components and/or to generate energy

reaction carry out the exergonic (spontaneous reactions) oxidation of nutrient

molecules to release free energy

2. Anabolism

or biosynthesis, in which biomolecules are synthesized from simpler components

free energy released by catabolic reaction is used to derive anabolic reaction which

is usually endergonic (non spontaneous reaction)

NB: Exergonic and endergonic reactions are often coupled through the intermediate

synthesis of a high energy compound, such as ATP

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Diagram shows theMetabolic Pathway

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Classification of organisms

Organisms can be classified in various ways:

First classification

Based on the nutritional requirements of an organism which reflect its source of freeenergy

Classified as autotrophs or heterotrophs

1. Autotrophs

Or self feeder, for prokaryotes, which can synthesize all their cellular

constituents from simple molecules such as H2O, CO2, NH3 and H2S

Subdivided into chemilithotrophs or photoautotrophs

a. Chemolithotrophs -- obtain free energy through oxidation ofinorganic

compounds, such as NH3, H2S and Fe2+

b. Photoautotrophs -- obtain free energy via photosynthesis: Inorganic cpds

CO2 (+light energy) carbohydrates (oxidized)free energy

2. Heterotrophs

Obtain free energy through the oxidation oforganic compounds such as

carbohydrates, lipids and proteins (synthesized by autotrophs:

chemolithotrophs or photoautotrops)

Organic compounds undergo oxidation release free energy

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Classification of organisms (continue..)

Second classification

Based on the identity of the oxidizing agent for nutrient breakdown

1. Obligate aerobeswhich include animals, must use O2

2. Anaerobesemploy oxidizing agents such as sulfate or nitrate

3. Obligate anaerobespoisoned by the presence of O2

4. Facultative anaerobessuch asE. coli can grow either in the presence or absence ofO2

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WHAT IS A METABOLIC PATHWAY?

Metabolic pathways

Are a series of connected enzymatic reactions that produce specific products;

many pathways are branched and interconnected

Metabolites

Refer to their reactants, intermediates and products

Metabolic Reaction

Over 2000 known, each catalyzed by a distinct enzyme

Types of enzymes and metabolites in a given cell

Vary with the identity of organisms, the cell type, its nutritional status and

developmental stage

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Catabolic and anabolic pathways are interconnected

Pathways can be classified as catabolic or anabolic

Catabolic pathway - complex metabolites are exergonically broken down into simpler

products, in many cases, into acetyl-CoA.

The free energy released is conserved by the synthesis of ATP from ADP+Pi or by the

reduction of the coenzyme NADP+ to NADPH

ATP and NADPH are the major free energy sources for anabolic reactions

Roles of ATP and NADPH in

metabolism

ATP and NADPH generated through

the degradation of complexmetabolites are the sources of free

energy for biosynthesis and other

reaction

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Metabolism

Complex metabolites such as

carbohydrates, proteins and lipidsare first degraded to their

monomeric units, chiefly glucose,

amino acids, fatty acids and

glycerols and then to a common

intermediate, acetyl-CoA

The acetyl-CoA is oxidized to CO2via the citric acid cycle with the

simultaneous reduction of NAD+

and FAD

Reoxidation of NADH and FADH2

by O2 during oxidative

phosphorylation yields H2O and

ATP

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Location of the Metabolic Pathway

Occur in specific location

Synthesis of metabolites in specific membrane-bounded compartment in

eukaryotic cells requires mechanism to transport these substances between

compartments, thus transport proteins are essential components of many

metabolic processes:

E.g., Transport protein is required to move ATP (generated in mitochondria) to

cytosol (where most of it is consumed)

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Location of the Metabolic Pathway (Continue..)

In multicellular organisms, compartmentation is carried a step further to the level of

tissues and organs, E.g.,

Liver -- synthesize glucose from non-carbohydrates precursor (gluconeogenesis) so as to

maintain a relatively constant level of glucose in the circulation whereas

Adipose tissue -- storage of triacylglycerols

Brain -- use glucose and ketone bodies for fuel

Muscle -- use ATP

Specialization in tissues and subcellular compartments is supported by the existenceof isozymes

Isozymes are enzymes that catalyzed the same reaction but are encoded by different genes

and have different kinetic or regulatory control

E.g., Lactate dehydrogenase (LDH)involves in the interconversion of pyruvate and

lactate; vertebrate posseses two homologs of this enzyme:

M type, function in tissues subject to anaeorobic condition such as skeletal tissue and

liver. In M-type, mainly function in the reduction of NADH of pyruvate to lactate

H type, function in tissues subject to aerobic condition such as heart muscle. In H

type, mainly function the reverse of the above reaction, i.e., lactate to pyruvate (A

blood test indicating the presence of H type LDH is diagnostic of a heart attack)

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Thermodynamic Consideration and the Control of Metabolic Flux

Given a biochemical reaction:

A + B C + D

Change in free energy (G) is related to the standard free energy (Go

)and theconcentration of the reactants,

G = Go + RTln

Where,

R = Gas constant, 8.3145 J.K-1.mol-1

T= Temperature in Kelvin, K (0oC=273.15K)

However when the reactants are present at values close to their equilibrium values,

Keq and G 0The reaction is said to be near-equilibrium reactions

Since G values are close to 0, they are reversible and this can be carried out by changing

ratio of products to reactants

In metabolic reactions, enzymes that catalyze near-equilibrium reactions tends to actquickly to restore the equilibrium constant

]B][A[

]D][C[

]B][A[

]D][C[

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Thermodynamic Consideration and the Control of Metabolic

Flux (continue)

Some metabolic reactions function far from equilibrium, therefore they are

irreversible,

A + B C + D

Enzymes that functions in reactions far from equilibrium has insufficient activity to

allow it to come to equilibrium and reactants accumulate in large excess of their

equilibrium amount, making

G

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Thermodynamic Consideration and the control of metabolic flux

(continue) Therefore, near-equilibrium reaction are freely reversible, whereas reactions that function far

from equilibrium serve as regulatory points and render metabolic pathway irreversible

Flux is the rate of flow of metabolite through a metabolic pathway To understand flux, we need to know which reactions are functioning near equilibrium and

which is functioning far from equilibrium

In the metabolic pathway, most enzymes operate near equilibrium and certain enzymes

operate far from equilibrium are strategically located to ensure the metabolic pathway

is irreversible. This has several implications:

1. Metabolic pathways are irreversible

A highly exergonic reaction (G

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Thermodynamic Consideration and the control of metabolic

flux (continue)

Flux through a metabolic pathway is controlled by regulating the activities of the

enzymes that catalyze its rate-determining steps by:1. Allosteric control by effectors

2. Covalent modification or enzyme interconversion e.g., by phosphorylation and

dephosphorylation

3. Substrate level

4. Genetic control

NB:

a. Mechanisms 1 to 3 can respond rapidly (within seconds or minute) to external stimuli

(short term control)

b. Mechanism 4 responds more slowly to changing conditions (within hours or days)

therefore regarded as long term control

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HIGH ENERGY COMPOUNDS IN THE METABOLIC

PATHWAY

In the metabolic pathway some high energy intermediates are synthesized They are like free energy currency and when they subsequently breakdown, the process

are always exergonic which can then drives any endergonic processes.

Such high energy compounds are, ATP and Phosphoryl Group

ATP: adenosine triphosphate

Occurs in all life form

Primary cellular energy currency

Consist of adenosine (adenine + ribose) sequentially linked to 3 phosphoryl gp

via a phosphoester bond followed by 2 phosphoanhydride bonds

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The structure of ATP

indicating its

relationship with ADP,AMP and adenosine

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ATP and Phosphoryl Group (continue.)

The free energy of the high energy compound ATP is made available through cleavage

of one or both of its phosphoanhydride bonds

This occur when either a phosphoryl gp is transferred to another cpd leaving ADP or anucleotidyl gp (AMP) is transferred leaving PPi. If the acceptor is water, the process is

called hydrolysis:

ATP + H2O ADP + Pi

ATP + H2O AMP + PPi

Most biological group-transfer reactions involve acceptors other than H2O

The free energy of hydrolysis of various phosphoryl compounds are known

Therefore we can calculate the energy of transfer of phosphoryl gps to other acceptors by

determining the difference in free energy of hydrolysis of the phosphoryl donor and

acceptor

The Gofor the hydrolysis of several phosphorylated cpds are as in Table in the next slide

f S i f i f

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Table of Standard Free Energies of Phosphate Hydrolysis of some

biological compounds

They are a measure of the tendency of phosphorylated compounds to transfer theirphosphoryl group to H2O

ATP has an intermediate phosphoryl group-transfer potential

Under standard conditions, compounds above ATP can spontaneously transfer a

phosphoryl group to ADP to form ATP, which can, in turn, spontaneously transfer a

phosphoryl group to the appropriate group to form the compounds listed below it

Group-transfer

potential is

intermediate forATP

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Why are ATP and some other phosphate compounds called high

energy compounds?

High energy phosphate compounds refer to compounds with standard free energy of

hydrolysis of more than -25 kJ.mol-1 (Refer to Table)

The phosphoanhydride groups are more destabilize than their hydrolysis products

There exists a competing resonances and charged repulsions between phosphoryl gp, thus

decreasing the stability of the phosphoanhydride bonds

Competing Resonance

Charge-charge repulsion

Phosphoanhydride

bonds

Hydrolysis products

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Sample calculation

Question:

Calculate the actual free energy of ATP hydrolysis at 37oC in a typical

cell, given [ATP]=3.0 mM, [ADP]=0.8 mM and [Pi]=4.0 mM

Answer:

ATP ADP + Pi

The actual free energy is:

G = Go+ RT ln

= -30.5 kJ.mol-1 + (8.3145 J.K-1)(310 K) ln (0.8 x 10-3 M)(4.0 x 10-3 M)(3.0 x 10-3 M)

= -30.5 kJ.mol-1 -17.6 kJ.mol-1

= -48.1 kJ.mol-1

]ATP[

]P][ADP[ i

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Coupled reactions

An exergonic rxn such as ATP or PPi hydrolysis can be coupled to endergonic reaction to

make it favourable

This is based on the additivity of free energy

E.g., Reaction 1: A + B C + D G1

Reaction 2: D + E F + G G2

If G1 0, the reaction will not occur spontaneously

If G2is sufficiently exergonic and G1+ G2 < 0, and although the equilibrium

concentration of D in Reaction 1 will be relatively small, it will be larger than that inReaction 2

As Reaction 2 converts D to products, Reaction 1 will operate forward to replenish the

equilibrium concentration of D

Therefore, the highly exergonic Reaction2 will drive the endergonic Reaction 1, and the two

reactions are said to be coupled through their common intermediate D, which proceedspontaneously

Summing up Reactions 1 and 2:

Reaction (1 + 2): A + B + E C + F + G G3

Where, G3= G1+ G2 < 0

As long as the overall pathway is exergonic, it will operate in the forward direction.

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Real examples of coupled reactions involving ATP

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Other phosphorylated Compounds

Other high energy compounds other than ATP are also essential in the metabolic

pathways

ATP is continually being hydrolyzed and regenerated

Previously we said that ATP drives endergonic reactions through the exergonic

processes of phosphoryl gp transfer and phosphoanhydride hydrolysis

ATP itself can be generated by coupling its formation to a more highly exergonic

metabolic processes

Table of Standard Free Energy of hydrolysis, Go, of some phosphate compounds

shows the position of ATP in relation to high energy and low energy phosphate

compounds

Substrate-level phosphorylation is the synthesis of ATP from ADP by direct transfer ofa phosphoryl group from another high energy compound

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Standard Free Energy of

hydrolysis, Goof

hydrolysis of some

phosphate compounds:

Position of ATP in relation

to high energy and low-

energy phosphate

compounds

Under standard conditions, the

high energy phosphate

compounds can spontaneously

transfer a phosphoryl group to

ADP to form ATP

ATP can spontaneously transfera phosphoryl group to the

appropriate group to form the

low energy phosphate

compounds

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Thioesters The common product of carbohydrate, lipid and protein catabolism, acetyl-CoA, is a

high energy thioester

Thioester bond which is a

high energy bond

Structure of Acetyl-CoA molecule

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Thioesters (continue.)

Coenzyme A- consist of B-mercaptoethylamine gp bonded thru an amide lingkage

to the vitamin panthothenic acid, which is attached to a 3-phosphoadenosine moiety

via a pyrophosphate bridge

The acetyl group is bonded as a thioester to the sulfhydryl portion of the B-

mercaptoetnoloamine group

Coenzyme A functions as a carrier of acetyl and other acyl group (The A of CoA

stands for acetylation)

Acetyl-CoA is a high energy compound because of the presence of a thioesterbond which is a high energy bond. The Go for the hydrolysis of its thioester bond

is -31.5kJ.mol-1, more exergonic than ATP hydrolysis and of ordinary esters

Other thioester compound is succinyl-CoA, present in the citric acid cycle

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OXIDATIONREDUCTION REACTIONS IN THE

METABOLIC PATHWAY

Oxidationlosing of electron (increase oxidation number e.g., Fe2+ to Fe3+)

Reductiongaining of electron (reduce oxidation number e.g., Fe3+ to Fe2+)

Oxidationreduction reactions are processes involving the transfer of electrons

Oxidationreduction reactions supply living things with most of their free energy

In photosynthesis, CO2 is reduced and H2O is oxidized to yield carbohydrates and O2 in a

process powered by light energy

In aerobic metabolism, the metabolites, carbohydrates and other organic compounds are

oxidized to CO2 to harvest the free energy in the form of ATP. The electrons are

transferred to molecular carriers which finally transferred the electrons to molecular

oxygen

In anaerobic metabolism, ATP is generated, although in lower yield through the

intramolecular oxidationreduction of various organic molecules, e.g. glycolysis or in

certain anaerobic bacteria, through the use of non-O2 oxidising agent such as sulfate or

nitrate

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OXIDATIONREDUCTION REACTIONS (Continue)

Oxidation and reduction reactions resemble other types of group-transfer reaction, only

that the group transferred in this case is electrons

Electrons (e

) are passed from an electron donor (reductant or reducing agent) to anelectron acceptor (oxidant or oxidizing agent)

Example:

Fe3+ + Cu+ Fe3+ + Cu2+

Cu+, the reductant is oxidized to Cu2+

Fe3+, the oxidant is reduced to Fe2+

Reduction oxidation reactions or redox reactions can be divided into two half-reactions or

redox couple

Fe3+ + e Fe2+ ------------ reduction

Cu+ Cu2+ + e ------------ oxidation

Fe3+ + Cu+ Fe2+ + Cu2+

Both the half-reactions occur simultaneously

The electrons are the two half-reactions intermediate

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Electrochemical Cells A half reaction consists of an electron donor and its conjugate electron acceptor

In the oxidative half-reaction, Cu+ is the electron donor and Cu2+ is its conjugate electron

acceptor

Together they are called conjugate redox pair

The twohalf reactions of a redox reaction each consisting of a conjugate redox pair can be

physically separated to form an electrochemical cell

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Each half-reaction takes place in its separate half-cell

The electron are passed between half-cells as an electric current in the wire connecting the

two electrodes

A salt bridge is necessary to complete the electrical circuit by providing a conduit for ions

to migrate and maintain electrical neutrality

The free energy of redox reaction can be determine by measuring the voltage between itstwo half-cells

Diagram of an electrochemical cell

i

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Redox Reaction

An+ox + Bred Ared + Bn+

ox

n = electrons per mole of reactants transferred from reductant (Bred) to oxidant

(An+ox)

The free energy is:

G = Go + RTln

Under reversible conditions,

G

= -w = -wel

Where, w = is non-pressure-volume work, in this case equivalent to wel, the electrical

work required to transfer the n moles of electrons thru the electrical potential difference,

, where are in volts (V), the number of joules (J) of work required to transfer 1

coulomb (C) of charge.

According to the Law of Electrostatics,wel = nF

Where

F, the faraday, is the electrical charge of 1 mol of electrons (1F =96,485 C.mol-1

=96,485 J.V-1.mol-1)

n= is the number of moles of electron transferred per mole of reactantconverted

[Ared][Bn+

ox]

[An+ox][Bred]

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Redox Reaction (Continue)

G = -w = -wel

Therefore,

G = -nF Substituting the above equation into the equation of change in free energy, yield the

Nernst equation,

= o ln where,

= the reduction potentialo = the standard reduction potential, the reduction potential when all componentsare in their standard states

= the electromotive force (emf), can be described as the electron pressure thatthe electrochemical cell exerts Positive result in negative G, in other words a positive indicates a spontaneous

reaction.

The Nernst equation relates the electromotive force of a redox reaction to the standard

reduction potentials and concentrations of the electron donors and acceptors.

RT

nF[Ared][B

n+ox]

[An+ox][Bred]

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Measurement of Reduction Potential

Any redox reaction can be divided into its component half-reaction:

An+ox + ne Ared

Bn+

ox + ne

Bred

Both half-reactions are written as reduction

In accordance with Nernst equation, these half-reaction can be assigned reduction

potentials, A and B:A= oA lnB= oB ln

For the overall redox reaction involving the half-reactions,

o = o(e- acceptor) - o(e- donor) If the reaction proceeds with A as the acceptor and B as the donor,

o = oA - oB Thus,

= A - B

RT

nF

[Ared]

[An+ox]

RT

nF[Bred]

[Bn+ox]

T bl h th St d d P t ti l f bi h i ll

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Table shows the Standard Potentials of some biochemically

important half-reactions

Slide 37

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Table shows the Standard Reduction Potential of Some

biochemically Important Half-Reactions

Features:

Oxidized form of a redox couple with a large positive standard reduction potential has

a high affinity for electrons and is a strong electron acceptor (oxidizing agent) and its

conjugate is a weak electron donor (reducing agent)

Electron flows spontaneously from the compound with the more negative reduction

potential (or low reduction potentials) to the compound with more positive reduction

potential (or high reduction potentials)

Electrons are transferred under standard conditions from the reduced products in any

half-reaction in the Table to the oxidized reactants of any half-reaction above it

Slide 38Sample calculation

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Sample calculation

Question:

Calculate G for the oxidation of NADH by FAD.

NADH NAD+ + H+ + 2e -----oxidation-lose electron, electron donor

FAD + 2H+ + 2e FADH2 -----reduction-gain electron, electron acceptor

Answer:

Combining the relevant half-reactions:

NADH + FAD + H+ NAD+ + FADH2

o = o(e-acceptor)o(e-donor)= o(FADH2/FAD)o(NADH/NAD+)= (0.219 V)(0.315 V)

= 0.096 V or J.C1

Since, G = -nF Therefore,Go= -nFo

=(2 mol e 1.mol 1 reactant) x (96,485 C.mol 1 e1)(0.096 J.C 1)

=18,500 J.mol 1 reactant

=18.5 kJ.mol 1 reactant

Slide 39

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Example of electron carriers in the metabolic pathway are:

NAD+ and FAD

NAD+ - nicotinamide adenine dinucleotide (phosphorylated counterparts NADP+)

FAD - flavin adenine dinucleotide

They are:

1. Nucleotide coenzymes (coenzymescomplex organic molecules, required by enzyme to

function)

2. The most widely occurring electron carriers in the metabolic pathway

3. The sites for reversible reduction during the oxidation of metabolites

Slide 40

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Reduction of NAD+ to NADH

Slide 41

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Reduction of FAD to FADH2

H. H.

Slide 42

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Electrons-transfers in biological system are important e.g., in

mitochondrial electron-transport chain

Electrons are passed from NADH along a series of electron acceptors of increasingreduction potential (including FAD and others listed in Table) to O2

ATP, a free energy currency, is generated from ADP and Pi by coupling its

synthesis to the above reactions

3 ATP molecules were generated from the oxidation of NADH to NAD+

NADH thus functions as an energy-rich electron transfer coenzyme

Slide 43

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EXPERIMENTAL APPROACHES TO THE STUDY OF

METABOLISM

A metabolic pathway can be understood at several levels:

1. In terms of the sequence of reactions by which a specific nutrients is converted to

end product and the energetics of these conversion

2. In terms of the mechanisms by which each intermediate is converted to its

successor. For such analysis, specific enzymes that catalyze each reaction need to

be isolated and characterized.

3. In terms of the control mechanisms that regulate the flow of metabolites thru the

pathway

Slide 44

EXPERIMENTAL APPROACHES TO THE STUDY OF

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EXPERIMENTAL APPROACHES TO THE STUDY OF

METABOLISM (continue)

To elucidate the metabolic pathway, many experimental approaches have been employed

including using metabolic inhibitors, growth studies and biochemical genetics

Metabolic inhibitors block pathways at specific enzymatic steps and the identification of

the resulting intermediates indicates the course of pathway

Mutation (naturally occurring genetic diseases) can be induced by mutagens, X-ray or

genetic engineering, may also result in the absence or inactivity of an enzyme

Modern genetic techniques make it possible to express genes in higher organisms(transgenic animals) or eliminate (knock out) a gene and study the effect of these effect on

metabolism

Isotopic labels can be incorporated into metabolites and allowed to enter a metabolic

system- their path may be traced from the distribution of label in the intermediates

NMR is a noninvasive technique that may be used to detect and study metabolites in vivo

Studies on isolated organs, tissue slices and subcellular organelles contributed to our

knowledge of the localization of metabolic pathway