Biochemistry Notes 3

HS COA

• COA+AMP

• Acyl CoA

Transport of Fatty Acyl CoA

• A transport system called the carnitine shuttle carries fatty acids into the mitochondria from the cytosol.

• Carnitine acyltransferase catalyzes the transfer of a fatty acyl group to the hydroxyl group of carnitine to produce fatty acyl carnitine.

• Fatty acyl carnitine then passes through the inner mitochondrial membrane into the matrix.

  $CH3(CH2)-CH2-CH2-C-S-COA + H-C-OH$
  Fatty acyl CoA

  $<br>CH2<br>|<br>N(CH3)3<br>|<br>COO<br>$
  Carnitine

  $<br>CH2 O<br>|<br>H-C-O-C-CH2-CH2-(CH2)-CH3$
  Fatty acyl carnitine

  $<br>CH2<br>|<br>N(CH3)3<br>|<br>COO</p></li><li><p>HS COA<br>$

• In the matrix, another carnitine acyltransferase

• Catalyzes the reverse reaction that transfers the fatty acyl group to CoA to reform fatty acyl CoA.

• Releases the carnitine & returns to the cytosol.

• Thus, the carnitine shuttle moves fatty acyl CoA from the cytosol into the matrix, where the fatty acid can undergo β oxidation.

• In the Carnitine shuttle system, fatty acids are activated & transported from the cytosol through the inner mitochondrial membrane into the matrix

Carnitine Shuttle System

• Fatty acids are linked to coenzyme A before they are oxidized.

• After being activated by linkage to CoA, the fatty acid is transferred to carnitine, a reaction catalyzed by carnitine acyltransferase I, for transport across the inner mitochondrial membrane. A translocase transports the acyl carnitine into the matrix of the mitochondria.

• In the mitochondria, carnitine acyltransferase II transfers the fatty acid to CoA. The fatty acyl CoA is now ready to be degraded.

  $H3C HO$

  $COA + H3C - N+ - CH3 + HS-COA$

  $H3C HO$

Diagram of Acyl-CoA and carnitine interaction through mitochondrial membrane

Numbering of Fatty Acid Carbon Atoms

• Fatty acid carbon atoms are usually numbered beginning w/ the carboxyl terminal C atom.

• C atoms 2 or 3 are also referred to as α, β, respectively

• The positions of double bonds are indicated w/ the symbol Δ, w/ the 1st atom of the double bond indicated by a superscript number.

• Δ9 indicates a double bond b/w C atoms 9 & 10.

• Fatty acids can also be numbered from the methyl C atom, which is called the omega (ω) carbon.

• total carbon : unsaturated cis/trans Δ/ω position of unsaturated carbons

• 18:4 cis-Δ6,9,12,15 18:4 cis-ω3,6,9,12

Different Types of Fatty Acids

• Saturated

• Aliphatic chain in fatty acid chain

• Even number of carbons (C16)

• Odd number of carbons (C17)

• Unsaturated

• Double bonds in fatty acid chain

• Odd double bond (18.4 cis-Δ6)

• Even double bonds (18:4 cis-Δ6,10)

Oxidation of Fatty Acids

• A large amount of energy is obtained when fatty acids undergo Oxidation in the mitochondria to acetyl CoA.

Beta oxidation (β oxidation)

• Removes 2-carbon segments containing the α & β carbon from the carboxyl end of the fatty acid.

β Oxidation

• A cycle in β oxidation:

• Produces an acetyl CoA & a fatty acid that is shorter by 2 carbons.

• Repeats until the original fatty acid is completely degraded to 2-carbon units that form acetyl CoA, which enters the citric acid cycle.

• Symbolic notation: β α

  $R-C-C-C$
  $H2 H2$
  $3 2 1$

Reaction 1: Beta (β) Oxidation of Fatty Acids

• Fatty acyl CoA undergoes beta (β) oxidation in a cycle of 4 reactions.

• In reaction 1:

  • Acyl CoA dehydrogenase catalyzes the transfer of H atoms from α & β carbons of the activated fatty acid.

  • A trans C=C bond is formed b/w α & β carbons, & the reduced coenzyme FADH2 is produced.

  $CH3(CH2)-CH2-CH2-C-S-CoA + FAD$
  Acyl CoA dehydrogenase
  $<br>CH3(CH2)n-C=C-C-S-CoA + FADH2$
  β α trans-Enoyl CoA

Reaction 2: Beta (β) Oxidation of Fatty Acids

• In reaction 2:

  • A hydration reaction catalyzed by enoyl CoA hydratase adds the components of H₂O to trans double bond.

  • A hydroxyl group (-OH) attaches to the β carbon of the fatty acid, & a H atom attaches to the α carbon.

  $CH3(CH2) C=C C-S-CoA + H₂O$
  Enoyl CoA hydratase
  $CH3(CH2) C-C-C-S-CoA$
  β α 3-Hydroxyacyl CoA

Reaction 3: Beta (β) Oxidation of Fatty Acids

• In reaction 3,

  • The secondary hydroxyl group on the β carbon (carbon 3) is oxidized by 3-hydroxyacyl CoA dehydrogenase to yield a ketone.

  • The H atoms removed in the oxidation are transferred to NAD+ to yield a β keto or 3-keto group & the reduced coenzyme NADH.

  $CH3 (CH2) C-C-C-S-CoA + NAD+$
  3-Hydroxyacyl CoA dehydrogenase
  $CH3-(CH2)n-C-C-C-S-COA +NADH + H+$
  β-Ketoacyl CoA B-Ketoacyl CoA

Reaction 4: Beta (β) Oxidation of Fatty Acids

• In reaction 4,

  • The Cα - Cβ bond is cleaved by β-ketoacyl CoA thiolase to yield a 2-carbon acetyl CoA & a new fatty acyl CoA that is shortened by 2 carbon atoms.

  • The shorter fatty acyl CoA repeats the 4 steps of the β-oxidation cycle until the original fatty acid is completely degraded to 2-carbon units of acetyl CoA.

  $CH3(CH2)n -C-C-C-S-CoA + HS-COA$
  B-Ketoacyl CoA thiolase

  $CH3(CH2)-2-C-S-CoA + CH3-C-S-COA$

Reaction Sequence for the Degradation of Fatty Acids

• Acyl CoA + FAD → trans-Δ²-Enoyl CoA + FADH2 (Oxidation)

• trans-Δ²-Enoyl CoA + H₂O → L-3-Hydroxyacyl CoA (Hydration)

• L-3-Hydroxyacyl CoA + NAD+ → 3-Ketoacyl CoA + NADH + H+ (Oxidation)

• 3-Ketoacyl CoA + HS-CoA → Acetyl CoA + Acyl CoA (shortened by two carbon atoms) (Thiolysis)

Fatty Acid Length Determines Cycle Repeats

• The # of carbons in a fatty acid determines the # of times the cycle repeats & the # of acetyl-CoA units it produces.

• The total # of times the cycle repeats is one fewer than the total # of acetyl groups it produces.

Beta (β) Oxidation of Capric Acid (C₁₀): Even Number of Carbons

• Capric acid (C10) undergoes 4 oxidation cycles that repeat reactions 1 to 4 & yield 5 acetyl CoA molecules, 4 NADH & 4 FADH2.

ATP from Fatty Acid Oxidation

• The energy yield from a fatty acid changes w/ the # of β oxidation cycles needed for its oxidation. Each cycle:

  • Requires an initial input of 2 ATP

  • Produces an NADH, an FADH₂, & one acetyl CoA

• NADH generates energy for the synthesis of 3 ATP.

• FADH2 provides energy for the synthesis of 2 ATP.

• Each β oxidation cycle produces 5 ATP.

The Krebs Cycle

• In addition, one molecule of ATP is generated.

• The energy tally from 1 molecule of pyruvic acid is: 3 NADH, 1 FADH₂, & 1 ATP

ATP Production from β Oxidation for Capric Acid (C10)

• Citric acid cycle: 5 acetyl CoA x 10 ATP = 50 ATP

Β oxidation:

• 4 NADH = 2.5 ATP NADH = 10 ATP

• 4 FADH₂ 1.5 ATP FADH₂ = 6 ATP

• Activation of capric acid: = -2 ATP

• Total = 64 ATP

Odd - Chain Fatty Acids Yield Propionyl CoA in the Final Thiolysis Step

• β oxidation of fatty acids w/ odd numbers of carbons generates propionyl CoA in the last thiolysis reaction.

• Propionyl CoA carboxylase, a biotin enzyme, adds a carbon to propionyl CoA to form methylmalonyl CoA.

• Succinyl CoA, a citric acid cycle component, is subsequently formed from methylmalonyl CoA by methylmalonyl CoA mutase, a vitamin B12-requiring enzyme.

  $H3C-C-COA$
  Propionyl CoA

Oxidation of Unsaturated Fatty Acids

• Some fats from our diets contain unsaturated fatty acids, which have ≥1 cis double bonds.

• An isomerase converts a cis double bond to a trans double bond b/w the α & β carbons so the fatty acid can undergo hydration.

• It forms a product that enters β oxidation @ reaction 2, so the energy released by the β oxidation of an unsaturated fatty acid is slightly less b/c no FADH2 is produced in that cycle.

The Degradation of Unsaturated & Odd-Chain Fatty Acids Requires Additional Steps

• An isomerase & a reductase are required for the oxidation of unsaturated fatty acids.

• β oxidation alone cannot degrade unsaturated fatty acids.

• When monounsaturated fatty acids such as palmitoleate are degraded by β oxidation, cis-Δ3-enoyl CoA is formed, which cannot be processed by acyl CoA dehydrogenase.

• Cis-Δ3 - enoyl CoA isomerase converts the double bond in trans-Δ2- enoyl CoA, a normal substrate for β oxidation

Oxidation of Linoleoyl CoA (Even Double Bonds)

Isomerase & Reductase Are Required for the Oxidation of Unsaturated Fatty Acids

• When polyunsaturated fatty acids are degraded by oxidation, cis-Δ3-enoyl CoA isomerase is also required. 2,4-dienoyl CoA is also generated but cannot be processed by normal enzymes.

• 2,4-dienoyl CoA is converted into trans-Δ3 - enoyl CoA by 2,4-dienoyl CoA reductase, &

• Unsaturated fatty acids w/ odd numbers of double bonds require only the isomerase. Even numbers of double bonds require both the isomerase & reductase

Citric Acid Cycle

• Step 1: Condensation

• Step 2: Isomerization

• Step 3: Decarboxylation, Oxidation

• Step 4: Oxidation, Decarboxylation

• Step 5: Hydrolysis

• Step 6: Oxidation

• Step 7: Hydration

• Step 8: Oxidation

Ketogenesis & Ketone Bodies

• If carbohydrates are not available,

  • Fatty acids break down to meet energy needs.

  • Acetyl CoA molecules combine to form ketone bodies

• When large quantities of fatty acids are degraded,

  • Too much acetyl CoA is produced

  • High levels of acetyl CoA accumulate in the liver

• Acetyl CoA molecules combine in a pathway known as ketogenesis to form compounds called ketone bodies

Ketone Bodies Are Another Fuel Source Derived from Fats

• Ketone - body synthesis takes place in the liver

• Ketone bodies—acetoacetate, D-3-hydroxybutarate, & acetone — are synthesized from acetyl CoA in liver mitochondria & secreted into the blood for use as a fuel by some tissues such as heart muscle

• $2 Acetyl CoA + H₂O → acetoacetate + 2 CoA + H+$

• D-3-hydroxybutyrate is formed upon the reduction of acetoacetate. Acetone is generated by the spontaneous decarboxylation of acetoacetate

• In tissues using ketone bodies, D-3-hydroxybutyrate is oxidized to acetoacetate, which is ultimately metabolized to 2 molecules of acetyl CoA

Formation of Ketone Bodies

Utilization of D-3-Hydroxybutyrate & Acetoacetate As a Fuel

Animals Cannot Convert Fatty Acids into Glucose

• Fats are converted into acetyl CoA, which is then processed by the citric acid cycle

• Oxaloacetate, a citric acid cycle intermediate, is a precursor to glucose

• However, acetyl CoA derived from fats cannot lead to the net synthesis of oxaloacetate or glucose b/c although 2 carbons enter the cycle when acetyl CoA condenses w/ oxaloacetate, 2 carbons are lost as CO₂ before oxaloacetate is regenerated

Step 1: Oxidation

• Fatty acid degradation consists of 4 steps that are repeated.

• Oxidation of the β carbon, catalyzed by acyl CoA dehydrogenase, generates trans-Δ² - enoyl CoA & FADH₂

Step 2: Hydration

• Hydration of trans-Δ²-enoyl CoA by enoyl CoA hydratase yields L-3-hydroxyacyl CoA

Step 3: Oxidation

• Oxidation of L-3-hydroxyacyl CoA by L-3-hydroxyacyl CoA dehydrogenase generates 3-ketoacyl CoA & NADH

Step 4: Cleavage

• Cleavage of the 3-ketoacyl CoA by β-ketothiolase forms acetyl CoA & a fatty acid chain 2 carbons shorter

The Complete Oxidation of Palmitate Yields 106 Molecules of ATP

• The reaction for one round of β oxidation is:

• $C-acyl CoA + FAD + NAD+ + H2O + CoA → C-2-acyl CoA + FADH2 + NADH + acetyl CoA + H+$

• The complete reaction for C16 palmitoyl CoA is:

• Palmitoyl CoA 7 FAD + 7 NAD+ + 7 CoA + 7 H₂O → 8 acetyl CoA 7 FADH2 + 7 NADH + 7 H+

• Processing of the products of the complete reaction by cellular respiration would generate 106 molecules of ATP

Nucleic Acids

• 2 types:
  * DNA
  * RNA
  * ATP

Nucleic Acids

• 2 types:

  • Deoxyribonucleic acid (DNA) may contain several million nucleotides & ribonucleic acid (RNA) may contain several thousand nucleotides

  • Both are unbranched polymers of repeating monomer units (nucleotides)

• Nucleic acids: store & transmit hereditary info

  • DNA: stores the info in genetic code

  • RNA: carries the info to the protein synthesizing machinery

Components of Nucleic Acids

• The general structure of a nucleotide includes:

  • Nitrogen-containing base

  • Sugar

  • Phosphate group

Pentose Sugars

• 5-carbon sugar

  • In RNA is ribose

  • In DNA is deoxyribose, w/o O atom on C2'

  • Has C atoms numbered w/ primes to distinguish them from atoms in the bases

Bases

• The bases in DNA & RNA are

  • Derivatives of the heterocyclic amines pyrimidine/purine

  • Pyrimidines w/ a single ring contains 2 N atoms. Each contains 2 N atoms.

  • Purines w/ 2 rings, each H+ acceptors in each base

Bases in DNA

• In DNA,

  • The purine bases w/ double rings are adenine (A) & guanine (G)

  • The pyrimidine bases w/ single rings are cytosine (C) & thymine (T)

Bases in RNA

• In RNA,

  • The purine bases w/ double rings are adenine (A) & guanine (G)

  • The pyrimidine bases w/ single rings are cytosine (C) & uracil (U)

Purines & Pyrimidines

Hydrogen Bonds Form Between an Electronegative Atom & Hydrogen

Nucleosides

• A nucleoside

  • Is composed of a nitrogen-containing base & a sugar, either ribose/deoxyribose

  • Has a base linked by a β - N-glycosidic bond to C1' of a sugar (ribose/deoxyribose)

Nucleotides

• A nucleotide has a phosphate group attached to the C5' — OH group of a nucleoside.

• The addition of a phosphate to a nucleoside forms a nucleotide

Nucleotides of DNA & RNA

Summary of the Components in DNA & RNA

TABLE 21.1 Components in DNA and RNA Components DNA RNA

• Bases : A, G, C, and T : A, G, C, and U

• Sugar : Deoxyribose : Ribose

• Nucleoside : Base + deoxyribose : Base + ribose

• Nucleotide : Base + deoxyribose + phosphate : Base ribose + phosphate

• Nucleic Acid : Polymer of deoxyribose nucleotides : Polymer of ribose nucleotides

TABLE 21.2 Nucleosides and Nucleotides in DNA and RNA Nucleosides Nucleotides Base DNA

• Adenine (A) : Deoxyadenosine (A) : Deoxyadenosine monophosphate (dAMP) Guanine (G)

• Deoxyguanosine (G) : Deoxyguanosine monophosphate (dGMP) Cytosine (C)

• Deoxycytidine (C) : Deoxycytidine monophosphate (dCMP) Thymine (T)

• Deoxythymidine (T) : Deoxythymidine monophosphate (dTMP) RNA Adenine (A)

• Adenosine (A) : Adenosine monophosphate (AMP) Guanine (G) : Guanosine (G)

• Guanosine monophosphate (GMP) Cytosine (C) : Cytidine (C) : Cytidine monophosphate (CMP)

• Uracil (U) : Uridine (U) : Uridine monophosphate (UMP)

Polymers Are Linked by Covalent Bonds

Primary Structure of Nucleic Acids

• In the primary structure of nucleic acids, each sugar in sugar-phosphate backbone is attached to base

• In the primary structure of nucleic acids,

  • The nucleotides are joined by phosphodiester bonds,

  • The 3'-OH group of the sugar in one nucleotide bonds to the phosphate group on the 5' C atom in the sugar of the next nucleotide

Base Sequence, Primary Structure

• Each nucleic acid has its own unique sequence of bases, which

  • Is known as its primary structure.

  • Carries genetic info

  • Is read from the sugar w/ the free 5' phosphate to the sugar group w/ the free 3'-OH

  • Is often written using letters of bases to represent the correct sequence

Backbones of DNA & RNA

• A base bound to a sugar is called a nucleoside. The nucleosides of DNA are deoxy adenosine, deoxyguanosine, deoxycytidine, & deoxythymidine. By convention, deoxythymidine, which rarely occurs in RNA, is simply called thymidine

• The nucleosides of RNA are adenosine, guanosine, cytidine, & uridine.

• In all cases, the C-1' of the sugar is attached to the N-9 of the purine or the N-1 of the pyrimidine

• A nucleotide is a nucleoside w/ ≥1 phosphoryl groups attached

• Nucleoside triphosphates are the building blocks of DNA & RNA

DNA Structure

• The building blocks of DNA are called nucleotides.

• One nucleotide is made of 3 important things:

• 5-Carbon sugar deoxyribose

• Phosphate

• Nitrogenous base

• There are 4 bases: adenine, guanine, cytosine, & thymine that pair together

  • A →T & G→C

RNA Structure

• The building blocks of RNA are nucleotides, just like DNA

• A nucleotide in RNA is made of:

  • 6-Carbon Sugar(ribose)

  • Phosphate

  • Nitrogen base

• There are 4: adenine, guanine, cytosine, & uracil

  • A→ U & G-C

The Double Helix

• This shows 2 strands of DNA in the form in which DNA is present w/in cells.

  • The sequence of nitrogenous bases is the code of DNA

  • Cytosine + Guanine

  • Adenine + Thymine

Deoxynucleotides Are More Stable

Base-Catalyzed Hydrolysis of RNA

• DNA & RNA differ w.r.t. the sugar found in their nucleotide monomer units (2-deoxyribose vs ribose)

• The phosphodiester bonds of RNA are susceptible to hydrolysis in basic solution due to the presence of the 2'-hydroxyl group of ribose

• In contrast, the phosphodiester bonds in DNA are much less susceptible b/c 2-deoxyribose lacks this group

• It is thought that DNA was selected over RNA as the preferred molecule for long-term storage of genetic info b/c it is less reactive molecule due to its containing 2-deoxyribose

DNA Double Helix

• In the model shown, the sugar - phosphate backbone is represented by a ribbon w/ hydrogen bonds b/w complementary base pairs.

Purines

• Adenine (A)

• Guanine (G)

Pyrimidines

• Cytosine (C)

• Thymine (T)

• Uracil (U) in RNA only

Complementary Base Pairs: A & T

• DNA contains complementary base pairs in which adenine is always linked by 2 H bonds to thymine (AT)

Complementary Base Pairs: G & C

• DNA contains complementary pairs in which guanine is always linked by 3 H bonds to cytosine (GC)

Double Helix of DNA

• The DNA structure is a double helix that

  • Consists of 2 strands of nucleotides that form a double helix structure like a spiral staircase

  • Has 2 strands held together by the H bonds b/w the bases AT & GC

  • Has bases along one strand that complement the bases along the other

DNA

• DNA, the genetic info that is passed from one generation to the next, is composed of 4 nucleotides w/ the bases A, G, C, & T

• DNA forms a double helix of 2 separate strands w/ complementary sequences

• During replication, the 2 strands unwind, each serving as a template for a new daughter double helix

DNA Structure

• General features of DNA as follows:

  • DNA molecules consist of 2 chains of opposite directionality—one strand runs in the 5'-to-3' direction & the other in the 3'-to-5' direction—intertwined to form a right-handed helix

  • The sugar-phosphate backbones are on the outside of the helix, whereas the bases are inside the helix

  • The bases are nearly perpendicular to the axis of the helix w/ adjacent bases separated by -3.4Å base separation

  • The helix is approximately 20 Å wide

An Axial View of DNA

A Nucleic Acid Consists of Bases Linked to a Sugar-Phosphate Backbone

• Nucleic acids are long, linear polymers constructed from 4 types of monomers.

  • Each monomer consists of a sugar, phosphate, & a base

  • The sequence of the bases is the info content of nucleic acid.

DNA & RNA Differ in the Sugar Component & One of the Bases

• The sugar component of deoxyribonucleic acid (DNA) is deoxyribose, a ribose in which the 2'-hydroxyl is replaced w/ hydrogen

• Ribonucleic acid (RNA) contains the sugar ribose

• The backbones of DNA & RNA consist of the sugars linked by phosphodiester bridges b/w the 3'-hydroxyl of one sugar & the 5'-hydroxyl of an adjacent sugar

• Bases are attached to carbon atom I' in the sugar.

• 2 of the bases are purines (adenine & guanine), & 2 are pyrimidines Cytosine & thymine / uracil】

DNA Molecules Are Very Long & Have Directionality

• Nucleic acid chains are presented by abbreviations such as pApGpCpT/pAGCT/AGCT

• Nucleic acid chains have directionality in that the 2 ends are different. One end has a free 5'-OH group or a 5'-OH group attached to a phosphoryl group & one end has a free hydroxyl attached to the 3' carbon of the sugar

• Nucleic acid chains are written in the 5'-to-3' direction

• DNA molecules can be extremely long, some consisting of >1 billion nucleotides in length

The Double Helix Is Stabilized by Hydrogen Bonds & Hydrophobic Effect

• Adenine always forms H bonds w/ thymine, whereas guanine forms H bonds w/cytosine

• The helix is stabilized by H bonds b/w base pairs as well as by hydrophobic interactions & van der Waals forces, called stacking forces, b/w adjacent bases

The Central Dogma

3 Major Hypotheses for DNA Replication

• Semiconservative: each strand serves as template for replication

• Conservative: intact double helix is copied in its entirety.

• Dispersive: replication results in hybrid's consisting of old & new DNA

Overview of Experiment

• Utilization of isotope

  • Chosen 15 N & 14 1 (common, lighter) & 1 (rare, heavier) key element of DNA Tabels to differentiate b/w parent & daughter DNA Technique of cesium chloride equilibrium density gradient centrifugation. Separation of molecules based on - Goal : detect whether new nitrogen atoms appear on I or both daughter strands methodology . Grew 1 generations E. . coli . :Changed medium to from this point forward all DNA replicated in this medium Periodically sampled the DNA grown in the medium . : (would be @ bottom of centrifuge)to means of equilibrium density gradient to compare the- Samples mixed w/cesium chloride, centrifuged & allowed to settle.

Zonal Centrifugation/Gradient Centrifugation

Meselson & Stahl Demonstrated That Replication Is Semiconservative

*DNA made up of only 15 N formed a single band band located @ midpoint b/w 15N & 1st .2nd replication generation had 2 bands (1 @ midpoint I @