biochemistry final (new material)

describe the differences between fatty acid catabolism and anabolism

catabolism

• produces acetyl-CoA

• produces reducing power (NADH, FADH₂)

• takes place in the mitochondria

anabolism

• requires acetyl-CoA and malonyl-CoA

• requires reducing power from NADPH

• takes place where NADPH levels are high (PPP) - in cytosol

where does anabolism of fatty acids occur

cytosol

• NADPH levels are high from PPP

why does anabolism of fatty acids require NADPH instead of NADH

because fatty acid synthesis takes place in the cytosol

• [NADPH] higher in cytosol

• [NADH] low in cytosol, high in mitochondria

fatty acids are built in several passes, processing one acetate unit at a time. where does each acetate unit come from?

• acetate comes from activated malonate in the form of malonyl-CoA

• each pass involves reduction of a carbonyl carbon to a methylene carbon

describe the general formation reaction for malonyl-CoA

acetyl CoA + HCO₃^- + ATP → malonyl-CoA + ADP + P_i

• enzyme: acetyl CoA carboxylase

• cofactor: biotin

what two enzymes are similar to acetyl-CoA carboxylase?

acetyl CoA carboxylase uses a two step reaction similar to carboxylations catalyzed by

• pyruvate carboxylase

  • gluconeogenesis

• propionyl CoA carboxylase

  • odd chain FA break down

describe the mechanism of the acetyl-CoA carboxylase reaction

1. use energy from ATP to add CO₂ to biotin in the biotin carboxylase site

   1. CO₂ is activated by attachment to N in ring of biotin

2. enzyme undergoes conformational change to carry CO₂ to transcarboxylase site

   1. swinging arm

3. CO₂ attaches to acetyl CoA and leaves active site

   1. malonyl CoA is produced

what is the overall goal of fatty acid synthesis

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

what are the four steps of FA synthesis

1. condensation

2. reduction

3. dehydration

4. reduction

describe step 1 of fatty acid synthesis

condensation of the growing chain with activated acetate

• make a C-C bond and release CO₂

describe step 2 of fatty acid synthesis

reduction of carbonyl to hydroxyl

• need oxidizing agent → uses NADPH, form NADP⁺

describe step 3 of fatty acid synthesis

dehydration of alcohol to trans-alkene

• remove H₂O from trans double bond between 𝛼 and 𝛽 carbons

describe step 4 of fatty acid synthesis

reduction of alkene to alkane

• reduce db again using NADPH

move lengthened acyl chain to KS so that malonyl CoA can bind ACP

how is the growing chain attached to the fatty acid synthase?

the growing chain is initially attached to the enzyme via a thioester linkage

what enzyme catalyzes fatty acid synthesis

fatty acid synthase (FAS)

what is the function of acyl carrier protein (ACP)

ACP serves as a shuttle in fatty acid synthesis. binds malonyl CoA

• contains a covalently attached prosthetic group

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

describe the structure of the ACP prosthetic group

prosthetic group contains pantothenic acid and an -SH group at the end where malonyl groups can bind

describe the function of the KS domain of FAS

make C-C bond, condensation

acetyl-CoA/growing chain attaches here

describe the function of the MAT domain of FAS

transferase, transfer growing chain to KS

describe the function of the DH domain of FAS

dehydration

describe the function of the ER domain of FAS

reduction (reductase)

describe the function of the KR domain of FAS

reduction (reductase)

write out the stoichiometry of synthesis of palmitate (16:0)

1. 7 acetyl-CoAs are carboxylated to make 7 malonyl-CoAs

7 acetyl-CoA + 7 CO₂ + 7 ATP → 7 malonyl-CoA + 7 ADP + 7 P_i

2. seven cycles of condensation, reduction, dehydration, and reduction

acetyl-CoA + 7 malonyl-CoA + 14 NADPH + 14H⁺ → palmitate + 7 CO₂ + 8 CoA + 14 NADP⁺ + 7 H₂O

why do acetyl groups need a shuttle?

acetyl groups are synthesized in the mitochondrial matrix. they are needed for fatty acid synthesis in the cytosol

describe the two shuttle systems that acetyl groups can use to get from mitochondria to cytosol

use citrate transporter

1. citrate + CoASH + ATP → oxaloacetate + ADP + P + acetyl CoA

   1. acetyl-CoA goes to FA synthesis

2. oxaloacetate + NADH + H → malate + NAD⁺

   1. malate can do malate-aspartate shuttle BUT most commonly it makes pyruvate

3. malate + NADP⁺ → NADPH + H + CO₂ + pyruvate

what enzyme is regulated in fatty acid synthesis

acetyl-CoA carboxylase (ACC)

• ACC catalyzes the rate limiting step

how is the ACC regulated?

allosterically regulated

• feedback-inhibited by palmitoyl-CoA

• activated by citrate

regulated by covalent modification

• inhibited when energy is needed

glucagon and epinephrine

how do glucagon and epinephrine regulate fatty acid synthesis?

low energy → do not consume lipids, phosphorylation cascade

lead to phosphorylation and inactivation of ACC

• ACC is inactive as phosphorylated monomers

• phosphorylate PKA to activate glycolysis

when dephosphorylated, ACC polymerizes into long, active filaments

• forms large active complex with lots of polymers

describe how fatty acid synthesis is regulated by phosphorylation

glucagon and epinephrine trigger phosphorylation

• phosphorylate ACC to inhibit fatty acid synthesis

• phosphorylate PKA to activate glycolysis

how does citrate regulate fatty acid synthesis

ACC is activated by citrate

• used citrate to shuttle acetyl CoA

• citrate in cytosol means high energy in cell to make fats

how does palmitoyl-CoA regulate fatty acid synthesis

feedback-inhibited by palmitoyl-CoA

• downstream inhibitor

describe reciprocal regulation of fatty acid metabolism using insulin

1. high blood sugar, increase insulin

2. dephosphorylate ACC to activate

3. perform FA synthesis, synthesize malonyl CoA

4. malonyl CoA inhibits fatty acid import into mitochondria

   1. FA cannot be broken down bc they are being made

• one of many ways to ensure that fat synthesis and oxidation don’t occur simultaneously

describe FA metabolism when blood sugar is low

• low BS, high glucagon

• ACC is phosphorylated and inactive

• low malonyl CoA allows entry of FA into matrix for break down

how do we store fats for energy

as triacylglycerols

what two molecules contain a glycerol backbone

TAGs and phospholipids

describe the structure of a TAG

3 fatty acids attached to a glycerol backbone

describe the structure of a phospholipid

2 fatty acid and a phosphate group attached to a glycerol backbone

what is the first step in making TAGs and phospholipids

make glycerol-3-phosphate first

why does triacylglycerol and phospholipid synthesis start the same way?

making glycerol-3-phosphate first tags it for TAG or phospholipid synthesis

describe the synthesis of the backbone of TAGs and phospholipids

most glycerol-3-phosphate comes from siphoning off dihydroxyacetone phosphate from glycolysis

• via glycerol-3-phosphate dehydrogenase (same enzyme from glycerol-3-P shuttle)

• use NADH

some glycerol-3-phosphate is made from glycerol

• via glycerol kinase

• use ATP

what is the precursor to TAGs and phospholipids

phosphatidic acid

what is the advantage of making phosphatidic acid

can be made into triacylglycerol or phospholipid

how is phosphatidic acid made

start with glycerol-3-phosphate

• 2 fatty acids attached by acyl transferases

• releases CoA

describe how phosphatidic acid can be modified to form TAGs

• phosphatidic acid phosphatase (lipin) removes the 3-phosphate from the phosphatidic acid

  • yields 1,2-diacylglycerol (DAG)

• the third carbon is then acetylated with a third fatty acid

  • uses acyl transferase

  • yields triacylglycerol

describe how phosphatidic acid can be modified to form phospholipids

attachment of head group (serine, choline, ethanolamine, etc.) to the phosphate group. yields glycerophospholipid

describe the steps of triacylglycerol synthesis

• DHAP + NADH + H⁺ → glycerol-3-phosphate + NAD⁺

  • glycerol-3-phosphate dehydrogenase

• glycerol-3-phosphate + 2 fatty acids → phosphatidic acid + 2 CoASH

  • acyl transferase

• phosphatidic acid → diacylglycerol

  • phosphatidic acid phosphatase

• diacylglycerol + fatty acid → triacylglycerol + CoASH

  • acyl transferase

describe the steps of phospholipid synthesis

• DHAP + NADH + H⁺ → glycerol-3-phosphate + NAD⁺

  • glycerol-3-phosphate dehydrogenase

• glycerol-3-phosphate + 2 fatty acids → phosphatidic acid + 2 CoASH

  • acyl transferase

• phosphatidic acid + head group → glycerophospholipid

how does insulin regulate triacylglycerol synthesis

insulin results in stimulation of triacylglycerol synthesis

• promotes synthesis is acetyl CoA and fatty acids

what does lack of insulin result in (lipid biosynthesis chapter)?

• increased lipolysis

• increased fatty acid oxidation

  • sometimes to ketones if citric acid cycle intermediates (OAA) that react with acetyl CoA are depleted

• failure to synthesize fatty acids

what is an eicosanoid?

• potent, short-range hormones made from arachidonate

• prostaglandins, leukotrienes, thromboxanes

• cause inflammation, pain, increased body temp

• released in response to injury

what are eicosaniods made from

made from arachidonate in the phospholipids of membranes

what enzyme is activated to release arachidonate

phospholipase A₂

what is PGH₂ synthase

a cyclooxygenase/peroxidase (COX) enzyme

describe eicosanoid synthesis

phospholipid containing arachidonate → arachidonate + phospholipid

• enzyme: phospholipase A₂

arachidonate + 2O₂ → PGG₂

• cyclooxygenase activity of COX

PGG₂ → PGH₂

• peroxidase activity of COX

PGH₂ → prostaglandins/thromboxanes

what do NSAIDs do

they inhibit cyclooxygenase (COX) activity

how does aspirin work?

aspirin (acetylsalicylate) is an irreversible inhibitor

• acetylates a serine in active site

• blocks active site in COX enzymes

how do ibuprofen and naproxen work

ibuprofen and naproxen are competitive inhibitors

• resemble substrate

• blocks active site in COX enzymes

what is cholesterol made from

made strictly from acetate units (starts as acetyl CoA)

provide the general steps for cholesterol synthesis

acetate (2C) → mevalonate (6C) → activated isoprene (5C) → squalene (30C) → cholesterol (27C)

describe the structure and function of mevalonate

composed of 3 acetate molecules

important intermediate in cholesterol synthesis

how are activated isoprenes created? what are they turned into?

reduce carbon on mevalonate and phosphorylate to make activated isoprene (5C)

6 isoprenes are needed to create squalene

what is squalene

the unfused (30C) intermediate of cholesterol. made from 6 isoprene molecules

what enzyme makes mevalonate? what is significant about this enzyme?

HMG-CoA reductase

first committed step of cholesterol synthesis, highly regulated

how do ATP levels regulate HMG-CoA reductase?

low [ATP] → high [AMP] → active AMPK → inhibit HMG-CoA reductase

how do insulin and glucagon regulate HMG-CoA reductase

insulin activates, glucagon inhibits

how does cholesterol regulate HMG-CoA reductase

high cholesterol → oxysterol → proteolysis of HMG-CoA reductase → inhibition

how does cholesterol travel through the body?

cholesterol and other lipids are carried on lipoprotein particles

describe the structure of lipoproteins

surface: protein (called apoliporotein) and phospholipid monolayer

interior: cholesterol, TAGs

rank the four classes of lipoprotein particles based on protein content

lowest: chylomicron

VLDL

LDL

highest: HDL

what does “density” refer to when talking about lipoprotein classes?

density refers to protein content

ex. HDL has more protein than LDL

what is the function of VLDL

transport TAGs to tissues

what is the function of LDL

transport cholesterol to tissues

how is cholesterol transported into tissue cells

• apolipoproteins bind to LDL receptor

• lipoproteins taken up by endocytosis

• internalized apolipoproteins are degraded in lysosomes, while cholesterol is used for biological function

note: number of receptors increase sin response to a need for cholesterol or steroid hormones

can cholesterol be metabolically degraded?

no! cholesterol cannot be metabolically degraded

• excess is returned to liver for ultimate excretion as bile to the intestine

what is the function of HDL

can participate in reverse cholesterol transport

• pick up cholesterol in extrahepatic tissues and carry to the liver for metabolism or excretion

describe the structure of HDL

more protein than lipid, contain relatively little cholesterol

describe how cardiovascular disease is multifactorial

very high LDL-cholesterol levels tend to correlate with atherosclerosis

• although many heart attack victims have normal cholesterol, and many people with high cholesterol do not have heart attacks

low HDL-cholesterol levels are associated with heart disease

what is familial hypercholesterolemia

• due to genetic mutation in LDL receptor

• impairs receptor-mediated uptake of cholesterol from LDL

• cholesterol accumulates in the blood/arteries

• rare, homozygous individuals can experience severe CVD/heart attacks as youths

how do statins work?

inhibit HMG-CoA reductase to lower cholesterol synthesis

• statins resemble mevalonate → competitive inhibitors of HMG-CoA reductase

how many amino acids can bacteria synthesize

can synthesize all 20

name the nonessential amino acids

• glutamate

• glutamine

• proline

• arginine (conditionally essential)

• serine

• glycine

• cysteine

• aspartate

• asparagine

• alanine

• tyrosine

name the essential amino acids

• valine

• histidine

• methionine

• isoleucine

• leucine

• lysine

• phenylalanine

• threonine

• tryptophan

what does it mean to be essential?

need to get it from the diet

what is the nitrogen source for amino acid synthesis

glutamine or glutamate

• 2 most plentiful amino acids

where do the carbon skeletons come from in amino acid synthesis

carbon skeletons derived from intermediates

• glycolysis

• citric acid cycle

• pentose phosphate pathway (bacteria only)

all amino acids in humans derive from one of four precursors. what are these precursors?

citric acid cycle

• 𝛼-ketoglutarate

• oxaloacetate

glycolysis

• pyruvate

• 3-phosphoglycerate

what amino acids are derived from 𝛼-ketoglutarate?

glutamate, glutamine, proline, arginine

how is glutamate synthesized

glutamate is derived from transamination of 𝛼-ketoglutarate

• need transaminase

how is arginine synthesized

synthesized from glutamate (via ornithine) in the urea cycle

• add guanidinium to ornithine, get nitrogen from glutamate

how is proline synthesized

proline can be synthesized from glutamate or from arginine

• ornithine is derived from glutamate, the urea cycle, or degradation of arginine

• ornithine aminotransferase converts ornithine to glutamate semialdehyde that cyclizes and converts to proline

  • deaminate ornithine to make something similar to glutamate

what amino acids are derived from 3-phosphoglycerate?

serine, glycine, cysteine

how is serine synthesized

oxidation → transamination → dephosphorylation

• 3 phosphoglycerate is oxidized

  • need reducing agent, NAD⁺

• add nitrogen from glutamate

• remove phosphate to yield serine

how is glycine synthesized

3-phosphoglycerate → serine → glycine

remove hydroxymethyl from serine to produce glycine

how is cysteine synthesized

3-phosphoglycerate → serine → cysteine

what amino acids are derived from oxaloacetate

aspartate and asparagine

how is aspartate synthesized

aspartate is formed from transamination of oxaloacetate

how is asparagine synthesized

oxaloacetate → asparatate → asparagine

asparagine is formed from transamidation of aspartate

what amino acids are derived from pyruvate

alanine

how is alanine synthesized

transamination of pyruvate forms alanine

• pyruvate + glutamate → 𝛼-ketoglutarate + alanine

how does consuming carbs lead to greater alanine production

metabolizing carbs: make pyruvate

• then pyruvate → alanine using glutamate as a nitrogen source