ADVANCED BIOCHEMISTRY
BISC 6310
FALL 2013
IUG, Fall 2013
Dr. Tarek Zaida
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COURSE TOPICS:
TEXTBOOK: BIOCHEMISTRY, 5TH ED. (LUBERT STRYER)
1.
2.
3.
4.
5.
6.
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Basic concepts of metabolism (ch. 8 & 14)
Glycolysis and gluconeogenesis (ch. 16)
The citric acid cycle (ch.17)
Oxidative phosphorylation (ch. 18)
Glycogen metabolism (ch. 21)
Fatty acids metabolism (ch. 22)
7. Protein turnover and amino acids metabolism (ch. 23)
8. Nucleotide biosynthesis (ch.25)
9. Biosynthesis of membrane lipids & steroids (ch. 26)
10. The Integration of metabolism (ch. 30)
11. Signal Transduction (ch.15)
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ACTIVITIES AND EXAMS
 Activities:
At the end of the term every student will present an oral
presentation of his/her choosing, covering one of the
medical-related disorders connected to any of the
course chapters
 You have to let me know which topic you have chosen
within 1 week from now, so that I may grant you the
permission to proceed with your research.
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 The evaluation of the presentation will be based on:
 Performance (oral)
 Content (oral & written)
 Time frame (limited to 10 min.)
 Note: Be ready to give me up your presentation electronically or
via email at : tzaida@iugaza.edu or tarekzaida@yahoo.com
 The time of the presentation should not exceed 10 minutes.
 Exams:
 Two exams will be held in the middle and at the end of the
semester.
 The material of the final exam will include up to 20% of the
chapters of the first term.
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 You will be given 4 Quizzes., the first , and second before midterm &
the third and fourth before final exam.
 Making-ups are not allowed neither for presentations nor for quizzes
nor for midterm ……………(STRICTLY ENFORCED)
 Class attendance is strongly recommended, But once you decide not
to come to class, its your responsibility to keep yourself posted and
updated with what has been done/ said during class.
 I will always welcome your suggestions as it is the best way to
improve the outcome of this class (you can talk to me in person or if
you feel more comfortable drop me an email)
 Office hours: male students Tue 2-3 pm
female students Sun: 2-3 pm
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GRADING:
Points
1 Presentation
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2 Different Electronic
Tools
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3 Quizzes
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4 Midterm exam
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5 Final exam
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Total
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100
METABOLISM
BASIC CONCEPTS & DESIGN
The quantitative study of cellular
energy transductions and the
chemical reactions underlying these
transductions.
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ENZYMES
Enzymes are the most sophisticated
biological catalysts known.
catalysts alter the rates of chemical reactions but
are neither formed nor consumed during the
reactions they catalyze.
Most enzymes are proteins. Some nucleic
acids exhibit enzymatic activities (e.g.,
rRNA).
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ENZYME CHARACTERISTICS:
RATE ENHANCEMENT
Enzymes significantly enhance the rates of
reactions:
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ENZYME CHARACTERISTICS:
TURNOVER NUMBERS
The “turnover number” is
used to rate the effeciency
of an enzyme.
It tells how many molecules
of reactant a molecule of
enzyme can convert to
product(s) per second.
How fast can an enzyme
produce products?
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ENZYME CHARACTERISTICS:
SPECIFICITY
 Enzymes can be very
specific.
 For example, proteolytic
enzymes help hydrolyze
peptide bonds in proteins.
 Trypsin is rather specific
 Thrombin is very specific
The specificity of an enzyme is due to the precise interaction
of substrate with the enzyme. This is a result of the intricate
three-dimensional structure of the enzyme protein.
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ENZYME CHARACTERISTICS:
REGULATION
Enzymes can enhance the rates of
reactions by many orders of magnitude.
A rate enhancement of 1017 means that what
would occur in 1 second with an enzyme’s help,
would otherwise require 31,710,000,000 years to
take place.
Thus, regulation of enzymatic activity is in a
sense, regulation of metabolism, or any
other cellular process
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ENZYME COFACTORS
 Many enzymes use
same cofactor
 Cofactors are split
into two groups:
 Metals
 Coenzymes (small
organic molecules)
Most vitamins are
coenzymes.
 When tightly bound
to enzyme, cofactor
=prosthetic group
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“Apoenzyme” + cofactor = “Holoenzyme”
ENZYMES – GIBBS FREE ENERGY
Gibb’s “Free Energy,” ΔG, determines the
spontaneity of a reaction:
ΔG must be negative for a reaction to occur
spontaneously
A system is at equilibrium and no net change can occur if
ΔG is zero
A reaction will not occur spontaneously if ΔG is positive;
to proceed, it must receive an input of free energy from
another source.
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DG = DHsystem - TDSsystem
 DG is the Gibbs free energy change, used to describe
energetics of biochemical reactions
 DH is the change in enthalpy, heat content of a system.
 DS is the change in entropy, a measure of the level of
randomness or disorder in a system.
 The total entropy of a system and its surroundings always
increases for a spontaneous process) {2nd Law}
 Entropy will increase only if: DG = DHsystem - TDSsystem < 0
 The free-energy change must be negative for a reaction to
be spontaneous
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 DG of a reaction depends only on free-energy of
products minus free-energy of reactants.
 DG of a reaction is independent of path (or molecular
mechanism) of the transformation
 DG provides no information about the rate of a reaction
 For the reaction: A + B  C + D
[C] [D]
ΔG  ΔG  RTln
[A] [B]
0'
 To determine DG, must consider nature of both
reactants and products as well as their concentrations
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FREE ENERGY & EQUILIBRIUM CONSTANT (K)
At equilibrium, DG = 0.
 0 = DGo’ + RTln([C][D]/[A][B])
 K’eq = [C][D]/[A][B]
DGo’ = - RTlnK’eq
An enzyme cannot alter
the equilibrium of a
chemical reaction.
This means, an enzyme
accelerates the forward and
reverse reactions by
precisely the same factor.
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ΔG0 '  RTlnK eq
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ENZYMES DECREASE ACTIVATION ENERGY
 A chemical reaction goes
through a transition state
with a higher DG than
either S of P
 Enzymes facilitate the
formation of the transition
state by decreasing DG‡
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ENZYME-SUBSTRATE COMPLEX
 For enzymes to function, they must come in contact with
the substrate.
 While in contact, they are referred to as the “enzyme-substrate
complex.”
 The combination of substrate and enzyme creates a new
reaction pathway, with a lowered transition-state energy
 More molecules have the required energy to reach the transition
state
 Catalytic power of enzymes is derived from the formation
of the transition states in enzyme-substrate (ES)
complexes
 The essence of catalysis is specific binding of the transition state
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ENZYME – ACTIVE SITE
Enzymes are often quite large compared to their
substrates. The relatively small region where the
substrate binds and catalysis takes place is called
the “active site.”
(e.g., human carbonic anhydrase:)
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ENZYME – ACTIVE SITE
 The active site is the region that binds the substrates (&
cofactors if any)
 It contains the residues that directly participate in the
making & breaking of bonds (these residues are called
catalytic groups)
 The interaction of the enzyme and substrate at the active
site promotes the formation of the transition state
 The active site is the region that most directly lowers DG‡
of the reaction - resulting in rate enhancement of the
reaction
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ENZYMES DIFFER WIDELY IN, STRUCTURE, SPECIFICITY, &
MODE OF CATALYSIS, YET, ACTIVE SITE HAVE COMMON
FEATURES:
 The active site is a 3-D cleft formed
by groups that come from different
parts of the amino acid sequence
 Water is usually excluded unless it
is a reactant.
 Substrates bind to enzymes by
multiple weak attractions
(electrostatic interactions,
hydrogen bonds, hydrophobic
interactions, etc.
 The specificity of binding depends
on the precisely defined
arrangement of atoms at the active
site
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ENZYME CLASSIFICATION
Enzymes are classified and named according to
the types of reactions they catalyze:
Proteolytic enzymes [such as trypsin] lyse protein
peptide bonds.
“ATPase” breaks down ATP
“ATP synthetase” synthesizes ATP
“Lactate dehydrogenase” oxidizes lactate, removing
two hydrogen atoms.
Such a wide variety of names can be confusing.
A better method was needed.
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ENZYME CLASSIFICATION
 The “Enzyme Commission” invented a systematic numbering system for
enzymes based upon these categories, with extensions for various subgroups.
e.g., nucleoside monophosphate kinase (transfers phosphates)
 EC 2.7.4.4. 2 = Transferase, 7 = phosphate transferred, 4=transferred to
another phosphate, 4 = acceptor
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LIVING ORGANISMS NEED ENERGY FOR:
1. Performing mechanical work
2. Active transport and maintaining
homeostasis
3. Synthesis of macromolecuels and
biochemicals.
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METABOLIC PATHWAYS
1. Catabolic pathways
2. Anabolic pathways
3. Amphibolic pathways that can be both
anabolic and catabolic depending on the
energy status in the cell.
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THE USEFUL FORMS OF ENERGY PRODUCED IN
CATABOLISM ARE EMPLOYED IN ANABOLISM
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A METABOLIC PATHWAY MUST SATISFY
MINIMALLY TWO CRITERIA
1. The reactions of the pathway must be
specific:
 This criterion is accomplished by enzymes
2. The entire set of reactions must be
thermodynamically favored.
 A reaction can occur spontaneously only if
DG, the change in free energy, is negative.
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AN IMPORTANT THERMODYNAMIC FACT
The overall free-energy change for a
chemically coupled series of reactions is
equal to the sum of the free energy
changes of the individual steps.
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The two reactions are catalyzed by the same enzyme
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ATP IS THE ENERGY CURRENCY IN
BIOLOGICAL SYSTEMS
The active form of ATP
is usually attached to
Mg2+ or Mn2+
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ATP IS THE ENERGY CURRENCY IN
BIOLOGICAL SYSTEMS
ATP is an energy-rich molecule because its
triphosphate unit contains two
phosphoanhydride bonds.
A large amount of free energy is liberated
when ATP is hydrolyzed to ADP and (Pi) or
to AMP and PPi.
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ATP IS THE ENERGY CURRENCY IN
BIOLOGICAL SYSTEMS
The precise DG°’ for these reactions
depends on:
The ionic strength of the medium
The concentrations of Mg2+ and other metal
ions.
Under typical cellular concentrations, the
actual DG for these hydrolyses is
approximately -12 kcal mol-1 (-50 kJ/mol).
ATP hydrolysis can be coupled to promote
unfavorable reactions.
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ATP HYDROLYSIS DRIVES METABOLISM BY SHIFTING THE
EQUILIBRIUM OF COUPLED REACTIONS
DG '0   RT ln K ' eq
DG ' 0
ln K ' eq  
RT
K ' eq  10
DG ' 0

RT
The equilibriu m constant at 25 o C is related to DG o ' by :
K ' eq  10

DG ' 0
1.36

[ B]eq
[ A]eq
 1.15  10 3
The molar ratio of B/A has to be lower than 1.15  10 3
fo the reaction to proceed.
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A CAN
BE CONVERTED INTO
COUPLED TO
ATP
B IF
THE REACTION IS
HYDROLYSIS
At pH 7.0 the equilibriu m constant of this coupled reaction is :
[ B]eq [ ADP ]eq [ Pi]eq
 DG o ' / 1.36
K ' eq 

 10
 10 3.3 / 1.36  2.67  10 2
[ A]eq
[ ATP]eq
At equilibriu m the ratio of B/A is given by :
[ B]eq
[ ATP]eq
 K ' eq
[ A]eq
[ ADP ]eq [ Pi]eq
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ATP IS AN ENERGY COUPLING AGENT:
[ATP]/[ADP ][Pi] ratio is maintained at high level in the cells (500M -1 )
[ B ]eq
[ A]eq
 2.67  10 2  500  1.34  10 5
This means that ATP hydrolysis enabels A to be converted into B until
the ratio [B]/[A] reaches a value of 1.34  10 5
The hydrolysis of an ATP molecule in a coupled
reaction changes the equilibrium by a factor of 108
(1.15 X10-3 compared to 1.34X105)
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A and B in the preceding example can be any
two different chemical species
A and B may represent activated and
unactivated conformations of a protein
phosphorylation with ATP may be a means of
conversion into an activated conformation.
Such a conformation can store free energy, which can
then be used to drive a thermodynamically
unfavorable reaction. (muscle contraction).
A and B may refer to the concentrations of an
ion or molecule on the outside and inside of a
cell, as in the active transport of a nutrient.
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THE STRUCTURAL BASIS OF THE HIGH
PHOSPHORYL TRANSFER POTENTIAL OF ATP
1.
Resonance stabilization:

2.
Electrostatic repulsion:


3.
At pH 7, the triphosphate unit of ATP carries about four
negative charges in close proximity.
The repulsion between them is reduced when ATP is
hydrolyzed.
Stabilization due to hydration:

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ADP and, particularly, Pi, have greater resonance stabilization
than does ATP.
Water can bind more effectively to ADP and Pi than it can to
the phosphoanhydride part of ATP, stabilizing the ADP and Pi
by hydration.
PHOSPHORYL TRANSFER POTENTIAL
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PHOSPHORYL TRANSFER POTENTIAL
It is significant that ATP has an intermediate
phosphoryl transfer potential among the
biologically important phosphorylated
molecules.
This intermediate position enables ATP to
function efficiently as a carrier of phosphoryl
groups.
If ATP had the highest potential then it wouldn’t
be formed.
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SOURCES OF ATP DURING EXERCISE
In resting muscle, [ATP] = 4 mM, [creatine phosphate] = 25 mM
[ATP] sufficient to sustain 1second of muscle contraction
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ATP-ADP CYCLE
 Only 100g of ATP in the body, turnover is
very high.
 This amount must be constantly recycled
every day. The ultimate source of energy
for constructing ATP is food; ATP is
simply the carrier and regulation-storage
unit of energy. The average daily intake of
2,500 food calories translates into a
turnover of a 180 kg of ATP
 Resting human consumes 40 kg of ATP
in 24 hours.
 Strenuous exertion: 0.5 kg / minute.
 2hr run: 60kg utilized
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CARBON FUELS- AN
ENERGY
IMPORTANT SOURCE OF
CELLULAR
The more reduced a carbon is, the more energy
its oxidation will give.
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Fats are a more efficient fuel source than
carbohydrates such as glucose due to the fact
that carbon in fats is more reduced.
Energy derived from carbon oxidation is used in:
 creating a high phosphoryl transfer potential
compound
 creating an ion gradient.
In both cases, the end point is the formation of ATP.
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THE
COMPLEXITY OF METABOLISM IS SIMPLIFIED BY
UNIFYING THEMES
 The use of activated
carriers is a recurring motif
in biochemistry:
- We’ve seen that phosphoryl
transfer can be used to drive
otherwise endergonic reactions,
- alter the energy of conformation
of a protein,
- or serve as a signal to alter the
activity of a protein. The
phosphoryl-group donor in all of
these reactions is ATP. So ATP is
an activated carrier of phosphoryl
groups because it is an exergonic
process.
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1. ACTIVATED ELECTRONS
OXIDATION.
CARRIERS FOR FUEL
Nicotinamide Adenine Dinucleotide
NAD+/NADH
The reactive part of NAD+ is its
nicotinamide ring, a pyridine
derivative synthesized from the
vitamin niacin.
Nicotinamide adenine dinucleotide
(NAD+): R = H
Nicotinamide adenine dinucleotide
phosphate (NADP+): R = PO3256
Oxidized
forms
In the oxidation of a substrate,
the nicotinamide ring of NAD+
accepts a hydrogen ion and
two electrons, which are
equivalent to a hydride ion and
becomes reduced.
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Flavin Adenine Dinucleotide
FAD/FADH2
This electron carrier consists
of a flavin mononucleotide
(FMN) unit (shown in blue)
and an AMP unit (shown in
black).
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FAD, like NAD+, can accept
two electrons. In doing so,
FAD, unlike NAD+, takes up
two protons.
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2. AN
ACTIVATED CARRIER OF ELECTRONS FOR
REDUCTIVE BIOSYNTHESIS
NADPH carries electrons in the same way as
NADH.
NADPH is used almost exclusively for
reductive biosyntheses, whereas NADH is
used primarily for the generation of ATP.
The extra phosphoryl group on NADPH is a
tag that enables enzymes to distinguish
between high-potential electrons to be used
in anabolism and those to be used in
catabolism.
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3. AN
ACTIVATED CARRIER OF TWO-CARBON
FRAGMENTS.
Coenzyme A, another central molecule in
metabolism, is a carrier of acyl groups
The terminal sulfhydryl group in CoA is the
reactive site.
Acyl groups are linked to CoA by thioester
bonds. The resulting derivative is called an
acyl CoA.
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An acyl group often linked to CoA is the
acetyl unit:
The DG° for the hydrolysis of acetyl CoA
has a large negative value:
Acetyl CoA has a high acetyl grouptransfer potential because transfer of the
acetyl group is exergonic.
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ACTIVATED CARRIERS
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A small set of carriers responsible for most interchanges
of activated groups in metabolism
NADH, NADPH, and FADH2 react slowly with O2
in the absence of a catalyst.
Likewise, ATP and acetyl CoA are hydrolyzed
slowly in the absence of a catalyst.
These molecules are kinetically quite stable in
the face of a large thermodynamic driving force
for reaction with O2 (in regard to the electron
carriers) and H2O (in regard to ATP and acetyl
CoA).
The kinetic stability of these molecules in the
absence of specific catalysts enables enzymes
to control the flow of free energy and reducing
power.
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KEY REACTIONS ARE REOCCURRING THROUGHOUT
METABOLISM
Types of chemical reactions in metabolism:
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1. OXIDATION-REDUCTION REACTIONS
Oxidation:
Loss of electrons
Loss of hydrogen
Gain of oxygen
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Reduction:
Gain of electrons
Gain of hydrogen
Loss of oxygen
2. LIGATION REACTIONS
Bond formation using free energy from ATP
cleavage.
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3. ISOMERIZATION REACTIONS
Rearrange particular atoms within the molecule.
Their role is often to prepare a molecule for
subsequent reactions such as the oxidationreduction.
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4. GROUP-TRANSFER REACTIONS
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5. HYDROLYTIC REACTIONS
Cleave bonds by the addition of water.
Hydrolysis is a common means employed to
break down large molecules.
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6. THE ADDITION OF FUNCTIONAL GROUPS TO
DOUBLE BONDS OR THE REMOVAL OF GROUPS
FORM DOUBLE BONDS
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IMPORTANT GENERAL PRINCIPLE OF
METABOLISM
Biosynthetic and degradative pathways are
almost always distinct.
It facilitates the control of metabolism.
Metabolic regulation and flexibility are
enhanced by compartmentalization:
For example, fatty acid oxidation takes place in
mitochondria, whereas fatty acid synthesis takes
place in the cytosol
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METABOLIC PROCESSES ARE REGULATED IN
THREE PRINCIPAL WAYS
1. The amounts of enzymes
2. Their catalytic activities
3. The accessibility of substrates
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1. THE AMOUNTS OF ENZYMES
Rate of enzyme synthesis:
Transcriptional regulation (ex. Lactose
upregulates b-Gal within minutes)
Translational regulation
Rate of enzyme degradation:
RNA degradation
Protein degradation
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2. THEIR CATALYTIC ACTIVITIES:
Reversible allosteric control:
Feed back inhibition of the first step of most
metabolic pathways by the pathway products.
Reversible covalent modification:
Phosphorylation-dephosphorylation.
Hormones like insulin and epinephrine trigger the
reversible modifications of key enzymes.
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3. THE ACCESSIBILITY OF SUBSTRATES
Controlling the flux of substrate
Transfer of substrate from one
compartment of the cell to the other.
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