Biochemistry Final

fatty acid biosynthesis

• while degradation of fatty acids takes place in mitochondria, the majority of fatty acid synthesis takes place in the cytosol

• intermediates covalently linked to acyl carrier protein

• activation of each acetyl CoA

• acetyl CoA +CO2 → Malonyl CoA

major repeating steps in the biosynthesis cycle

• condensation

• reduction

• dehydration

• reduction

fatty acid synthesis

1. the enzymes of fatty acid synthesis are packaged together in a complex called fatty acid synthase (FAS)

2. the product of FAS action is palmitic acid (16:0) → can’t produce anything longer

3. modifications of this primary FA lead to other longer (and shorter) FA and unsaturated FA

4. the fatty acid molecule is synthesized 2 carbons at a time

5. FA synthesis begins from the methyl end and proceeds toward the carboxylic acid end. thus, C16 and C15 are added first and C2 and C1 are added last

6. C15 and C16 are derived directly from acetyl CoA. For further stepwise 2-carbon extensions, acetyl COA is first activated to malonyl CoA, a 3-carbon compound, by the addition of a CO2

citrate shuttle

• FAs are synthesized in the cytoplasm from acetyl CoA

• acetyl CoA generated from pyruvate by the action of PDH and by beta-oxidation of fatty acids in the mitochondria

• for fatty acid biosynthesis, acetyl CoA has to be transported from the mitochondria to the cytoplasm. this is done via a shuttle system called the citrate shuttle

• acetyl CoA reacts with oxaloacetate to give citrate. A tricarboxylate translocase transports citrate from mitochondria to cytosol

• in the cytosol, citrate is cleaved back to oxaloacetate and acetyl CoA. this reaction is catalyzed by ATP-citrate lyase and requires the hydrolysis of one molecule of ATP

citrate shuttle steps

• oxaloacetate is converted back to pyruvate for re-entry into mitochondria

• 1. oxaloacetate + NADH + H+ → malate + NAD+

  • catalyzed by cytosolic malate dehydrogenase

• 2. malate + NADP+ → pyruvate + CO2 + NADPH

  • catalyzed by malic enzyme

• pyruvate translocase transports pyruvate into mitochondria

• pyruvate is converted to oxaloacetate by pyruvate carboxylase with coupled hydrolysis of one ATP (uses one ATP)

  • pyruvate + ATP + CO2 + H2O → oxaloacetate + ADP + Pi

• Net reaction: NADP+ + NADH + H+ + ATP + H2O → NADPH + NAD+ + ADP + Pi

• thus, transport of acetyl CoA to cytosol requires expense of one ATP and conversion of one NADH to NADPH

net reaction of citrate shuttle

• NADP+ + NADH + H+ + ATP + H2O → NADPH + NAD+ + ADP + Pi

what is the committed step of fatty acid synthesis

• formation of malonyl CoA

formation of malonyl CoA (is the committed step of fatty acid synthesis)

• carboxylation of acetyl CoA to malonyl CoA → catalyzed by acetyl CoA carboxylase I

• acetyl CoA is combined with HCO3-, the formation of CO2, in aqueous solutions

• this is irreversible

what is the input to fatty acid synthesis?

• acetyl CoA

  • a two-carbon compound which is carboxylated to three carbon compound malonyl-CoA

  • ATP-dependent carboxylation provides energy input

  • the CO2 is lost later during condensation with the growing fatty acid

  • the spontaneous decarboxylation drives the condensation reaction

what catalyzes the 2-step reaction by which acetyl-CoA is carboxylated to form malonyl CoA?

• acetyl-CoA carboxylase

• as with other carboxylation reactions, the enzyme prosthetic group is biotin

• ATP-dependent carboxylation of the biotin, carried out at one active site 1, is followed by transfer of the carboxyl group to acetyl CoA at a second active site 2

what is the precursor for fatty acid synthesis?

acetyle CoA

the conversion of acetyl CoA to malonyl CoA is the rate limiting step in the fatty acid synthesis

true

in what compartment does the de novo fatty acid synthesis occur?

cytosol

the overall reaction (acetyl → malonyl CoA) which is spontaneous

• HCO3- + ATP + acetyl CoA → ADP + Pi + malonyl CoA

fatty acid synthase (FAS)

• a polypeptide chain with multiple domains, each with distinct enzyme activities required for fatty acid biosynthesis

• acyl carrier protein (ACP): for fatty acid biosynthesis, the activator is a protein called the acyl carrier protein (ACP). it is part of the FAS complex. the acyl groups get anchored to the CoA group of ACP by a thioester linkage

• condensing enzyme/beta-ketoacyl synthase (cys-SH). also part of FAS, CE has a cysteine SH that participates in thioester linkage with the carboxylate group of the fatty acid

• during FA biosynthesis, the growing FA chain alternates between Cys-SH and ACP-SH (important)

acyl carrier protein (ACP)

• for fatty acid biosynthesis, the activator is a protein called the acyl carrier protein (ACP). it is part of the FAS complex. the acyl groups get anchored to the CoA group of ACP by a thioester linkage

what serves as a shuttle in fatty acid synthesis?

• acyl carrier protein (ACP)

acyl carrier protein serves as a shuttle in fatty acid synthesis

• contains a covalently attached prosthetic group 4’-phosphopantetheine

• flexible arm to tether acyl chain while carrying intermediates from one enzyme subunit to the next

• delivers acetate (in the first step) or malonate (in all the next steps) to the fatty acid synthase

• shuttles the growing chain from one active site to another during the four step reaction

step-wise reactions (biosynthesis)

• the acetyl group gets transferred from CoA to ACP by acetyl CoA-ACP translocase

• the acetyl (acyl) group next gets transferred to the cys-SH arm of FAS complex

• next, the malonyl group gets transferred from CoA to ACP by malonyl CoA ACP translocase. this results in both arms of FAS occupied forming acylmalonyl-ACP

• the COO group of malonyl ACP is removed as CO2, the acetyl group (C16 and C15) gets transferred to the alpha carbon of malonyl ACP

• this results in 3-keto acyl ACP

• 3- keto group is converted to a CH2 by a series of reactions reverse to FA beta-oxidation

• namely…

  • 1. reduction to hydroxyl group. enz. 3-keto acyl ACP reductase

  • 2. dehydration to form a 2,3 double bond and enz. 3-hydroxy acyl ACP dehydratase

  • 3. a second reduction to remove the double bond. enz: enoyl ACP reductase

• both reduction reactions require the reduced cofactor NADPH. this is generated by the hexose monophosphate pathway of phosphogluconate pathway and during the citrate shuttle

repeat cycles for chain elongation

*the result of the first cycle of fatty acid biosynthesis is a four carbon chain associated to the ACP arm

*this chain gets transferred to the cys-SH arm

*a new malonyl CoA is introduced on the ACP arm

*the reactions proceed as before. for each cycle the acyl group transferred to the alpha carbon of malonyl CoA is c-carbons longer the previous cycle

→ *at the end of 7 cycles a 16 carbon chain is attached to the ACP arm (palmitoyl ACP)

*the C16 unit is hydrolyzed from ACP yielding free palmitate

*net reaction: acetyl CoA + 7 malonyl CoA + 14 NADPH + 14 H+ → palmitate + 7 CO2 + 8 CoA + 14 NADP+ + 6H2O

goal of fatty acid synthesis

*attach acetate unit (2-carbon) from malonyl-CoA to a growing chain and then reduce it

FA biosynthesis involved cycles of four enzyme-catalyzed steps:

*condensation of the growing chain with activated acetate

*reduction of carbonyl to hydroxyl

*dehydration of alcohol to transalkene

*reduction of alkene to alkane

enzymes of fatty acid synthesis

• condensation with acetate

  • beta-ketoacyl-ACP synthase (KS)

  reduction of carbonyl to hydroxyl

  • beta-ketoacyl-ACP reductase (KR)

• dehydration of alcohol to alkene

  • beta-hydroxyacyl-ACP dehydratase (DH)

• reduction of alkene to alkane

  • enoyl-ACP reductase (ER)

• chain transfer/charging

  • malonyl/acetyl-CoA ACP transferase

stoichiometry of synthesis of palmitate (16:0)

*7 acetyl CoAs are carboxylated to make 7 malonyl CoAs… using ATP

→ 7 acetyl CoA + 7CO2 + 7 ATP = 7 malonyl CoA + 7 ADP + 7 Pi

*seven cycles of condensation, reduction, dehydration, and reduction…using NADPH to reduce the beta-keto group and trans-double bond

→ acetyl CoA + 7 malonyl CoA + 14 NADPH + 14 H+ = palmitate (16-carbons) + 7 CO2 + 8 CoA + 14 NADP+ + 6 H2O

how to make longer fatty acids (ex. C18)

• obtained by addition of one or more additional C2 fragments by a different enzyme system (elongases)

• mammals cannot synthesize linolate or a-linolenate (essential FA)

what do biosynthesis and beta oxidation have in common?

• *acetyl CoA

  • *it is the end product of each spiral of beta oxidation

  • *fatty acids are synthesized two carbon atoms at a time

    • the source of the two carbon atoms is the acetyl group of acetyl CoA

desaturation of fatty acid by fatty acyl-CoA desaturase

• O2 accepts four electrons from two substrates

• two electrons come from saturated fatty acid

• two electrons come from ferrous state of cytochrome b5

what desaturates fatty acids?

• fatty acyl-CoA desaturase

regulation of fatty acid synthesis

• acetyl CoA carboxylase reaction is a rate limiting step in FA biosynthesis

• → palmitoyl-CoA: allosteric inhibitor (feedback inhibition);

• → citrate (ATP and acetyl-CoA high in mitochondria): allosteric activator

• → covalent modification: phosphorylation: inhibit

• → dephosphorylation: polymerization and activation

do fatty acid oxidation and synthesis occur at the same time

• no, malonyl CoA inhibits carnitine acyltransferase I and then shuts down FA oxidation

plants and fatty acid biosynthesis

• acetyl CoA carboxylase is activated by the changes in Mg+2 and pH (increase) that accompany illumination

regulation of FA metabolism

• → the two processes of beta oxidation and FA synthesis are coordinately regulated

• → three hormonal signals determine the state of FA metabolism. glucagon and epinephrine inhibit FA synthesis and favor oxidation, whereas insulin is anti-lipolytic and stimulates FA biosynthesis

• → the mechanism of hormonal regulation is covalent phosphorylation of acetyl CoA carboxylase, the rate-limiting step of FA biosynthesis

• → acetyl CoA carboxylase is inhibited by phosphorylation. phosphorylated acetyl CoA carboxylase can regain partial activity by allosterically binding citrate

how does glucagon and epinephrine effect fatty acid synthesis and oxidation?

• inhibit FA synthesis and favor oxidation

how does insulin effect fatty acid synthesis and oxidation?

• anti-lipolytic and stimulates FA biosynthesis

allosteric regulation of FA metabolism

→ acetyl CoA carboxylase is the rate limiting step of FA biosynthesis. it is allosterically inhibited by palmitoyl CoA and activated by citrate

→ palmitoyl CoA also inhibits the citrate shuttle and thus slows down FA biosynthesis

→ malonyl CoA and acetyl CoA inhibit beta oxidation

→ acetyl CoA inhibits the final step of beta oxidation catalyzed by 3-ketoacyl thiolase

→ malonyl CoA inhibits the transport of acyl CoA to mitochondria via inhibition of carnitine-acyl transferase I

→ additionally, ATP and NADH also inhibit beta oxidation

common features of membranes

• → sheet-like flexible structure, 30-100 A (3-10nm) thick

• → main structure is composed of two leaflets of lipids (bilayer)

• → protein molecules span the lipid bilayer

• → electrically polarized (inside negative ~ -60mV)

• → asymmetric

structural lipids in membranes (polar)

• → contain polar head groups and nonpolar tails (usually attached fatty acids)

• → diversification can come from:

  • → modifying a different backbone

  • → changing the fatty acids

  • → modifying the head group

• → the properties of head groups determine the surface properties of membranes

• → different organisms have different membrane lipid compositions

• → different tissues have different membrane lipid compositions

membrane bilayers are asymmetric

• → two leaflets have different lipid compositions

• → outer leaflet is often more positively charged

• → phosphatidylserine outside has a special meaning:

  • → platelets: activates blood clotting

  • → other cells: marks the cell for destruction

why is phosphatidylserine important for bilayer and cells?

• → when membranes lose asymmetry, the cell dies

• → it is most commonly found in the inner leaflet

  • → when it moves to the outer leaflet it leads to cell death

    • → platelets: activates blood clotting

    • → other cells: marks the cell for destruction

organisms can adjust the membrane composition

• → membrane fluidity is determined mainly by the fatty acid composition

• → more fluid membranes require shorter and more unsaturated fatty acids

• → at higher temperatures cells need more saturated fatty acids to maintain integrity

• → at lower temperatures cells need more unsaturated fatty acids to maintain fluidity

more fluid membranes require

• shorter and more unsaturated fatty acids

at higher temperatures cells need

• more saturated fatty acids to maintain integrity

at lower temperatures cells need

• more unsaturated fatty acids to maintain fluidity

membrane fusion

• → membranes can fuse with each other without losing continuity

• → it can be spontaneous or protein mediated

• → examples of protein-mediated fusion are:

  • → entry of influenza virus into the host cell

  • → release of neurotransmitters at nerve synapses

  • → liposome drug delivery

caveolin forces membrane curvature

• feature is a caveola

  • → gives place for many proteins to come together and interact with each other

  • → a lot of cholesterol is found in them

membrane rafts

• → lipid rafts

  • → contain clusters of glycosphingolipids with longer-than-usual tails

  • → are more ordered

  • → contain specific doubly or triply acylated proteins

  • → allow segregation of proteins in the membrane

protein channels, gates, pumps

• * nutrient transport

• * ion channels

• * neurotransmitters (eg: serotonin reuptake protein)

enzymes (membrane)

• → lipid biosynthesis (some acyltransferase)

• → ATP synthesis (F0F1 ATPase/ATP synthase)

• → receptor tyrosine kinases

functions of proteins in membranes: the basis of bio-signaling

• transport

• enzymatic activity

• signal transduction

• cell-cell recognition

• intercellular joining

• attachment to the cytoskeleton and extracellular matrix (ECM)

receptors

• → detecting signals from outside

  • → light (opsin)

  • → hormones (insulin receptor)

  • → neurotransmitters (acetylcholine receptor)

  • → pheromones (taste and smell receptors)

membrane proteins

• channels, gates, pumps

• enzymes

• receptors

• cell adhesion

amino acids in membrane protein cluster

• → transmembrane segments are predominantly hydrophobic

• → tyr and trp cluster at nonpolar/polar interface

• → charged residues (lys, arg, glu, asp) are found almost exclusively in the aqueous phases

types of membrane proteins

• → peripheral membrane proteins

• → integral membrane proteins

• → lipid-linked membrane proteins

peripheral membrane proteins

• → associate with the polar head groups of membranes (doesn’t go through the membrane)

• → relatively loosely associated with membrane

  • → through ionic interactions with the lipids or aqueous domains of integral membrane proteins

• → removed by disrupting ionic interactions either with high salt or change in pH

lipid-linked membrane proteins

• → reversible process

• → allows targeting of proteins

• → some, such as GPI anchors are found only on the outer face of the plasma membrane

integral membrane proteins

• → span the entire membrane

• → have asymmetry like the membrane

  • → different domains in different compartments

• → tightly associated with membrane

  • → hydrophobic stretches in the protein interact with the hydrophobic regions of the membrane

• → removed by detergents that disrupt the membrane

• → integral membrane proteins are purified to lipids

six types of integral membrane proteins

type 1

type 2

type 3

type 4

type 5

type 6

types of cell adhesion molecules

• → homophilic binding

• → heterophilic binding

cell adhesion molecules (CAMs)

• → can be divided into four major families

  • → the cadherin superfamily

  • → the selectins

  • → the immunoglobulin superfamily

  • → the integrins

cadherins

• → have a short transmembrane domain and a relatively long extracellular domain containing four cadherin repeats (EC1-EC4), each of which contains calcium binding sites

• → cadherins interact with specific cytoplasmic proteins e.g. catenin’s (alpha, beta, and gamma), through which they link the actin microfilament

• → the binding of cadherins to the catenin’s is crucial for cadherin function

integrins

• → transmembrane receptors that are the bridges for cell-cell and cell-extracellular matrix (ECM) interactions. when triggered, integrins trigger che