advanced biochemistry bisc 6310 fall 2013

ADVANCED BIOCHEMISTRY

BISC 6310 FALL 2013

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IUG, Fall 2013Dr. Tarek Zaida

COURSE TOPICS:TEXTBOOK: BIOCHEMISTRY, 5TH ED. (LUBERT STRYER)1. Basic concepts of metabolism (ch. 8 & 14)2. Glycolysis and gluconeogenesis (ch. 16)3. The citric acid cycle (ch.17)4. Oxidative phosphorylation (ch. 18)5. Glycogen metabolism (ch. 21)6. Fatty acids metabolism (ch. 22)

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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:

Points1 Presentation 7

2 Different Electronic Tools

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3 Quizzes 16

4 Midterm exam 20

5 Final exam

Total

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100

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METABOLISMBASIC 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 ENHANCEMENTEnzymes 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

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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.

ENZYME CHARACTERISTICS:REGULATIONEnzymes 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

14 “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

spontaneouslyA system is at equilibrium and no net change can occur if

ΔG is zeroA 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 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

DG = DHsystem - TDSsystem

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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

To determine DG, must consider nature of both reactants and products as well as their concentrations

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[B][A][D][C]RTlnΔGΔG 0

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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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eq0 RTlnK'ΔG

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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

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 work2. Active transport and maintaining

homeostasis3. 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 enzymes2. 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 FACTThe 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

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The active form of ATP is usually attached to Mg2+ or Mn2+

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ATP IS THE ENERGY CURRENCY IN BIOLOGICAL SYSTEMSATP 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 mediumThe 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

RTG

eq

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A CAN BE CONVERTED INTO B IF THE REACTION IS COUPLED TO ATP HYDROLYSIS

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ATP IS AN ENERGY COUPLING AGENT:

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of value a reaches [B]/[A] ratio theuntil B into converted be to Aenabels hydrolysis that ATP means This

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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 proteinphosphorylation 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:

ADP and, particularly, Pi, have greater resonance stabilization than does ATP.

2. Electrostatic repulsion: 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. 3. Stabilization due to hydration:

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

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In resting muscle, [ATP] = 4 mM, [creatine phosphate] = 25 mM[ATP] sufficient to sustain 1second of muscle contraction

ATP-ADP CYCLE

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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

CARBON FUELS- AN IMPORTANT SOURCE OF CELLULAR ENERGYThe 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 CARRIERS FOR FUEL OXIDATION.

The reactive part of NAD+ is its nicotinamide ring, a pyridine derivative synthesized from the vitamin niacin.

Nicotinamide adenine dinucleotide (NAD+): R = HNicotinamide adenine dinucleotidephosphate (NADP+): R = PO3

2-

Nicotinamide Adenine DinucleotideNAD+/NADH

Oxidizedforms

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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 DinucleotideFAD/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 group-transfer potential because transfer of the acetyl group is exergonic.

ACTIVATED CARRIERS

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A small set of carriers responsible for most interchangesof activated groups in metabolism

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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

Reduction:Gain of electronsGain of hydrogenLoss of oxygen

Oxidized

Reduced

Oxidation:Loss of electronsLoss of hydrogenGain of oxygen

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2. LIGATION REACTIONS

Bond formation using free energy from ATP cleavage.

3. ISOMERIZATION REACTIONS

Rearrange particular atoms within the molecule.Their role is often to prepare a molecule for

subsequent reactions such as the oxidation-reduction.

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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 METABOLISMBiosynthetic 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 WAYS1. The amounts of enzymes2. Their catalytic activities3. 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 degradationProtein degradation

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 substrateTransfer of substrate from one

compartment of the cell to the other.

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