mbb 321 final

How malonyl co-A formed

Transcarboxylase transfers activated CO2 from biotin to acetyl-CoA

• done by acetyl coa carboxylase

• is the committed step in fatty acid synthesis

location of fatty acid synthesis

cytosol

how acetyl coA exported from mitochondrial matrix to cytosol

shuttle system

• citrate formed in matrix from acetyl coA + oxaloacetate from CAC

  • if too much citrate, its transported out to cytosol

• in cytosol, citrate → acetyl coA + oxaloacetate

  • using citrate lyase + 1ATP

• oxaloacetate → malate

  • using malate dehydrogenase to return to matrix

  • some malate → pyruvate by malic enzyme thru oxidative decarboxylating

  • generates NADPH for FA synthesis

• pyruvate → oxaloacetate back in mitochondria

3 functional regions of acetyl coa

1. biotin carrier protein

2. biotin carboxylase

   • activates CO2 by attaching it to nitrogen in biotin ring in an ATP dependent reaction

3. transcarboxylase

   • transfers activated CO2 from biotin to acetyl coA producing malonyl coA

acetyl coA carboxylase

single protein w 2 identical polypeptide chains

• acetyl coa carboxylase activity depends on polymerization: filamentous form = active; monomers/dimers = inactive

• citrate as allosteric activator of ACC shifts equilibrium toward active filamentous form

fatty acid synthase complex components

6 domains have enzymatic activity

• 1 domain carries ACP: acyl carrier protein

How fatty acids synthesized w thioester linkages

focus on activation by thioester linkages + where thioesters located

• malonyl + acetyl activated thru thioester linkage to FAS

• malonyl group attached to ACP (acyl carrier protein)

• acyl group attached to thioester on other domain

• in next round of FAS, 4C fatty acyl group moves to thioester on KS domain where acetyl was in first round

fatty acid synthesis steps

1. condensation - thru decarboxylation

2. reduction

3. dehydration

4. reduction

CRDR

Where carbons come from in fatty acids and during synthesis

during condensation:

• 4C carried on ACP

• methyl C from acetyl coA

• other C from malonyl coA

at end of first round of FAS complex:

• 4C fatty acyl group (butyryl-ACP)

• in next round of FAS, 4C fatty acyl group moves to thioester on KS domain where acetyl was in first round

• THEN NEW malonyl group added to ACP

during second round:

• condense 4C butyryl group + 3C malonyl

  • uses decarboxylation

• leaves 6C on ACP

• last 3 steps result in 6C fatty acyl group attached to ACP (hexanoyl-ACP)

TE (thioesterase domain)

has hydrolytic activity

• releases free palmitate from ACP thru hydrolysis

FAS stopping elongation

continues till 7 cycles make 16C saturated palmitoyl bound to ACP

• stops cuz free palmitate released from ACP thru TE hydrolysing

• less longer fatty acids also formed

• in coconut/palm, termination earlier, 90% stops 8-14C

Where fatty acids are elongated, focus on numbering!!

2C added to carboxyl end

• shifts numbering bc count starts at carboxyl end

• ex: 1C → 3C

Where mammals can de-saturate

cannot introduce double bonds into fatty acid chain beyond delta9

• when linoleic acid (18:2C), used as substrates for other elongation + desaturation rxns

• arachidonic acid (20C) produced is used for phospholipid synthesis

  • phospholipids then hydrolysed for eicosanoid biosynthesis (signalling regulating molecules)

insulin - regulation of fatty acid synthesis in animal cells

stimulates fatty acid synthesis

• helps glucose into cells

• increase fluctuations thru glycolysis and pyruvate dehydrogenase rxn

  • gives acetyl CoA for FAS

fatty acyl coA - regulation of fatty acid synthesis in animal cells

provides inhibition feedback of FAS to prevent polymerisation/activation of acetyl-coA carboxylase

• inhibition occurs cuz acetyl-coA carboxylase role to convert acetylcoa→malonylcoa is rate limiting step in biosynthesis of fatty acids

therefore important site of regulation!!

malonyl coA - regulation of fatty acid synthesis in animal cells

blocks beta-oxidation, preventing fatty acids from entering mitochondria by inhibiting carnitine acyltransferase I

citrate - regulation of fatty acid synthesis in animal cells

acts as allosteric activator

• helps acetyl-coA carboxylase tend toward active filamentous form

NADPH - regulation of fatty acid synthesis in animal cells

its availability controls fatty acid synthesis bc it comes from:

• transport of citrate out of mitochondria

• pentose phosphate pathway (PPP)

location of fatty acid elongation and desaturation

smooth ER

salvage pathway of phosphatidylcholine synthesis

• fatty acid binding proteins (FABPs) help move fatty acids

• fatty acids activated by adding coA

• 2 fatty acids + glycerol-3-phosphate = phosphatidic acid

  • done with acyl transferases

• phosphate removed from phosphatidic acid = DAG (diacylglycerol)

  • done with phosphatase

• DAG (giacylglycerol + (choline+P) from CDP choline = phosphatidylcholine

• phosphatidylcholine transferred into luminal leaflet w flippase

alternate pathway of phosphatidylcholine synthesis

3 successive methylations of phosphatidyethanolamine = phosphatidylcholine

• methyls from adomet (transfers carbons in most reduced state)

• ATP cleaved = inorganic triphosphate (PPPi) + adenosyl moiety linked to methionine sulfur

  • adenosyl moiety + methionine sulfur = unstable sulfonium ion

synthesis of methionine + S adenosylmethionine in activated methyl cycle

bc of unstable sulfonium, methyl group VERY reactive, more than tetrahydrofolate methyl

• s-adenosyl-methionine + methyl from adomet = s-adenosyl-homocysteine

• s-adenosylhomocysteine = homocysteine + adenosine

• homocysteine + methyl from H4folate = methionine

  • uses methionine synthase

similarity btw methionine synthase vs methylmalonyl coA → succinyl coA

both only known coenzyme B12 dependent rxns in mammals

metabolic fates of acetyl coA

1. oxidation to CO2 in CAC

2. biosynthesis of fatty acids

3. biosynthesis cholesterol

4. ketogenesis

location of cholesterol synthesis

in liver

where C in cholesterol from

derived from acetate

mevalonate formation

1. 2x acetyl-CoA → acetoacetyl-CoA

   • uses thiolase

2. acetoacetyl-CoA + acetyl-coA → b-hydroxy-b-methylglutaryl-coA (HMG-CoA)

   • uses HMG-coA synthase

IRREVERSIBLE FROM HERE

3. HMG-CoA + 4e- from NADPH + H+ → mevalonate

   • uses HMG-CoA reductase

   • is integral membrane protein of smooth ER

   • primary regulatory step of cholesterol synthesis!

similarities btw ketogenesis + synthesis of mevalonate

both use HMG-CoA synthase for:

• acetoacetyl-coA → b-hydroxy-b-methylglutaryl-coA (HMG-CoA)

differences btw ketogenesis + synthesis of mevalonate

ketogenesis:

• uses HMG-CoA LYASE to convert

• b-hydroxy-b-methylglutaryl-CoA (HMG-CoA) into acetoacetate

mevalonate

• uses HMG-CoA REDUCTASE to convert

• b-hydroxy-b-methylglutaryl-CoA (HMG-CoA) into mevalonate

4 stages of choelsterol synthesis

1. formation of mevalonate

2. conversion of mevalonate to 2 active isoprenes

3. condensation of activated isoprene units to form squalene

4. squalene to cholesterol

conversion of mevalonate to 2 active isoprenes

• mevalonate activated by 3 phosphorylation from ATP

• 3rd phosphorylation @ C3 preps molecule for decarboxylation

• when phosphate + carboxyl leave, double bond made!

• 2x isopentyl pyrophosphate produced

isopentenyl pyrophosphate

active isoprene

• precursor for many molecules

• yields dimethylallyl pyrophosphate (other activated isoprene)

HMG CoA reductase

committed step

• primary regulatory step in cholesterol biosynthesis

condensation of activated isoprene units to form squalene

• isopentenyl pyrophosphate + dimethylallyl pyrophosphate → geranyl pyrophosphate

  • condensated thru 1 pyrophosphate displaced

• isopentyl pyrophosphate + geranyl pyrophosphate → farnesyl pyrophosphate

• farnesyl pyrophosphate + 2e- from NADPH + H → squalene

squalene to cholesterol

• squalene → squalene 2,3-epoxide → lanosterol

• lanosterol → cholesterol

  • thru 20 rxns w double bond rxns + 3 demethylations

cholesterol biosynthesis regulation

HMG-coA reductase (committed rxn enzyme) target for regulation

• regulated w insulin + glucagon transcriptionally

• dietary cholesterol suppresses inner cholesterol synthesis

• rate of inner cholesterol synthesis controlled by rate of cholesterol by cells + dietary intake

regulatory effects of internalised cholesterol

inner cholesterol synthesis suppression thru:

1. inhibit HMG-CoA reductase

   • suppress HMG transcription

   • hurry up degrading HMG

2. activate acyl-coA-cholesterol-acyltransferase (ACAT)

   • synthesises cholesterol esters from cholesterol + long chain acyl-CoA

3. decrease mRNA for LDL receptor

   1. ensures cholesterols dont enter cell

regulate HMG-CoA reductase thru suppression + activation of gene transcription

controlled by small family of proteins called sterol regulatory element binding proteins (SREPB)

• high SREBP if low [sterol], helps increase cholesterol synthesis

• when new SREBPs embedded in ER membrane, only soluble amino terminal domain is activator

  • BUT can only activate cholesterol transcription in nucleus

• if low SREBP cuz high [sterol], its bound to SCAP (SCREBP cleavage activating proteins)

  • SCAP binds cholesterol + other proteins

effects of internalised cholesterol in cell

elevated blood cholesterol levels cuz extracellular cholesterol accumulates + has nowhere to go

where drugs come from

1. plants

2. human derived proteins/steroids

3. fungi/bacteria

4. synthetic chemicals

5. recombinant proteins

what Kd

strength of interactions btw drug + target

• lower Kd = stronger interaction/affinity

• kd = [receptor]*[ligand]/[receptor+ligand}

• kd = [free ligand] where half receptor bind sites occupied (x @ y/2)

how Kd affected by natural substrate (physiological conditions)

target molecule binds normal ligand, so drugs need to compete

• amount drug needed depends on [normal substrate] to compete

• therefore, more normal substrate, more drug needed to be effective

what Ki

inhibition constant, apparent dissociation constant

• kd^app = kd * (1 + [S]/Km)

what EC50 and EC90

EC = Effective concentration

EC50 = [drug] needed for 50% of max response

EC90 = [drug] needed for %90 of max response

• ex. for an antibiotic, EC90 = concentration needed to kill 90% of bacteria exposed

what agonist + function

binds receptor to evoke response

what antagonist + function

drug binding to receptor without activating, blocking agonists from binding

what reuptake inhibitor + function

blocks neurons from reabsorbing neurotransmitters resulting in more neurotransmitters available

• ex. SSRIs as antidepressants, making serotonin more available

ADME properties of a drug

properties needed for drugs to be EFFECTIVE

• A = absorption

• D = distribution

• M = metabolism

• E = excretion

what first pass metabolism

the amount of an ORALLY administered drug thats metabolised by liver the first time it gets there

• can significantly alter bioavailability of drug

bioavailability

fraction of drug available in blood supply after administrated (systemic circulation)

what oral bioavailability

subcategory of bioavailability

• fraction of drug available in blood supply after drug ingested

• after drug survives acidic gut, its absorbed into bloodstream bc not soluble

• enough of the drug must then reach target compartment

role of p450s in drug metabolism

oxidises + conjugates foreign compounds (xenobiotics) to excrete them

• usually in liver

how drug metabolised affect bioavailability

as drugs metabolised, their [] decreases, so drug needs to be administered more frequently

• gets oxidised first cuz adding OH gives opportunity to attach smthn else

• half life = [drug] decreases to ½ of its value over time

  • important for determining how often drug needed

effect of drug interactions w other drugs

may control properties of proteins different from but related to target molecule

• accidentally modulates protein unrelated to intended target

• ex. antiviral drug against viral protease inhibits other protease that good for blood pressure

what therapeutic index of drug

ratio of dose of a compound required to elicit a toxic response in 1/s of its test subjects [LD50 lethal dose] to a comparable measure of its effective dose (EC50)

• way to describe toxicity of drug

• ex. if TI = 10, lethality is high when 10x effective dose administered

how calculate therapeutic index of drug

lethal dose/ recommended dose

• ex. lethal = 10,000, recommended = 3000, 10,000/3000 = 3.3

SPMs (specialised pro-resolving mediators)

involved in antinflammatory responses

source of arachadonic acid

derived from phospholipids (phosphatidylinositol)

3 pathways for arachidonic from phospholipids

1. phospholipids → lysophospholipid + arachidonic acid

   • using phospholipase A2

2. phospholipid → 1,2-diacylglycerol(DAG) → phosphatidic acid → phosphatidic acid + arachidonic acid

   • using phospholipase C, diacylglycerol kinase, phospholipase A2

3. phospholipid → 1,2-diacylglycerol(DAG) → monoacylglycerol + arachidonic acid

   • using phospholipase C, diacylglycerol lipase

3 stages of prostaglandin + thromboxane synthesis

Stage 1: Release of arachidonic acid

• Membrane phospholipids are cut by phospholipase A₂ (PLA₂)

• This releases arachidonic acid from the cell membrane

Stage 2: Conversion to PGH₂ (common intermediate)

• Arachidonic acid is converted by cyclooxygenase (COX-1 / COX-2)

• Produces PGH₂, the key intermediate

Stage 3: Formation of specific products

• PGH₂ is converted into different eicosanoids depending on the cell:

  • Prostaglandins (PGs)

  • Thromboxanes (TXs)

2 stages of PGHs synthase reactions

1. cycloxygenase activity

   • Adds oxygen to arachidonic acid

   • Forms PGG₂ (a hydroperoxide intermediate)

2. peroxidase activity

   • Reduces PGG₂

   • Converts it into PGH₂

how NSAIDS inhibit PGH2 synthase

work as anti-inflammatory agents treating pain + inflammation

NSAIDS blocks access of arachidonic acid binding to cox1/cox2

• aspirin thru acetylating, aspirin inhibits both equally

diff btw cox1 vs. cox2

cox1

• 70kD protein always expressed in mammals + supports prostaglandin synthesis to maintain tissue/organ homeostasis

cox2

• 74kD protein w 60% homology w cox2, inducible and only expressed in tissues in response to inflammatory stimuli, and responsible for high prostaglandin causing inflammation, PAIN, fever

• overexpressed in cancer and associated with tumour growth + resistance to conventional treatments

benefit of cox2 specific inhibitor

cox 2 active site slightly larger than cox1, so can be cox2 specific

• doesnt have side effects of other NSAIDS

role of low doses of aspirin preventing heart attack + stroke

thromboxane synthase converts PGH2 → thromboxane A2

• thromboxanes induces blood vessel constricting + platelet aggregating + blood clotting

aspirin blocks cox1 so less thromboxanes = less clotting

source of amino acids that get metabolised

dietary + tissue protein

metabolic circumstances for amino acids to get metabolised

1. when amino acids released during normal protein turnover, and not needed for new protein synthesis

2. in protein rich diet, when amino acids ingested exceed whats needed for protein synthesis (cuz aminos cant be stored)

3. during starvation or diabetes mellitus when carbs are either unavailable or not properly utilised, cellular proteins used as fuel

ultimately, if aminos not used for biosynthesis, they come ammonia to be excreted

amino acids in carnivores vs plants

carnivores - can obtain 90% energy requirements from amino acid oxidation

plants - dont rlly metabolise amino acids for energy

• carbs from CO2 + H2O instead

• amino acid catabolism in plants generates metabolites for other biosynthetic pathways

• [amino acids] regulated to meet biosynthesis requirements for growth

conditionally essential amino acids

body can make it only if theres enough precursor

transamination rxn

alpha-amino group transferred to alpha-carbon of alpha-ketoglutarate generating glutamate

• purpose is to collect aminos as L-glutamate for biosynthetic pathway or to excrete them

• first step in catabolism after reaching liver

PLP info

pyridoxal phosphate (coenzyme form of vitamin B6)

• acts as intermediate carrier of amino groups at active site of aminotransferases

• PLP bound to enzyme thru schiff base

• new schiff base formed btw alpha amino group and incoming amino acid and PLP

• proton lost leaving behind free e- pair on alpha-carbon

• amino group of first incoming amino is part of pyridoxamine phosphate

• rest of amino acid leaves in form of alpha-keto acid

• reverse direction to complete rxn w diff alpha-keto acid coming in and new amino acid leaving

mechanism of PLP rxn (ping-pong rxn)

• first substrate reacts (incoming amino acid donates its amino group to pyridoxal phosphate)

• first product leaves as alpha-keto acid of first substrate

• second substrate reacts (incoming alpha-keto acid diff from one that just left)

• second product leaves (amino acid formed from second substrate alpha-keto acid and amino group from first substrate amino acid)

role of aminotransferases in diagnosis of liver disease

if aminotransferase serum levels found in liver (SGPT + SGOT) high, liver cells are damaged cuz enzymes leaking into blood

• helps diagnose liver injury/damage caused by drug toxicity or infection

role of troponin T in diagnosis of heart disease

troponin T test - troponin helps regulate muscle contraction

• troponin T in heart is structurally different from troponin in other muscle

glutamine synthetase rxn

1. NH3 + glutamate + ATP → y-glutamyl phosphate intermediate

2. y-glutamyl phosphate + ammonia → glutamine + Pi

bc NH3 toxic to humans, glutamine brought into mitochondrial matrix then released as free ammonia

glucose alanine cycle

if enough pyruvate:

• pyruvate + amino group → alanine

• alanine in liver → transaminated into pyruvate, amino on glutamate

• glutamate in mitochondria processed in urea cycle, pyruvate for gluconeogenesis

• lactate from muscle to liver for gluconeogenesis

difference btw transamination vs. deamination

transamination (move)

• ex. Glutamate + NH₃ → Glutamine

deamination (delete)

• ex. glutamate → α-ketoglutarate + NH₃

carbamoyl phsophate

formed in mitochondrial matrix

• ATP dependant

• CO2 produced in respiration during formation

• catalysed by carbamoyl phosphate synthetase I

• ONE amino group carried by it!!

• is then considered an activated compound to enter urea cycle

urea cycle in detail (substrates, enzymes, products)

1. ornithine + carbamoyl phosphate → citrulline

   • enzyme = ornithine transcarbamolase

   • moves into cytosol of hepatocyte

2. citruline + amino group from aspartate → arginosuccinate

   • enzyme = arginosuccinate synthetase

   • occurs in cytosol, 2*2ATP = 4ATP used so far

3. arginosuccinate → arginine + fumarate

   • enzyme = arginosuccinase

   • arginine has both aminos + stays in cytosol

   • fumarate moves into mitochondrial matrix

4. arginine + H2O → urea released + ornithine regenerated

   • enzyme = arginase

fumarate fate after urea cycle

enters mitochondrial matrix for CAC

• generates NADH to help synthesise 3 ATP

• 3 ATP slightly offsets 4 ATP used in urea cycle

• aspartate also gets regenerated

3 ways carbons are carried in metabolic rxns

1. biotin - transfers carbons as CO2 (most oxidised)

2. s-adenosyl methionine (adomet) - transfers carbons as methyl groups (most reduced)

3. tetrahydrofolate - carries 1-carbon groups in intermediate states of oxidation, sometimes methyl groups

   • folic acid is its precursor

where carbons carried on tetrahydrofolate

held on nitrogen at position 5 + 10, or bridged between

• C come from beta carbon of serine

synthesis of tetrahydrofolate

• folate + 2H → dihydrofolate

• dihydrofolate → tetrahydrofolate

  • H’s from NADPH + H

  • dihydrofolate reductase converts thru reductions!

relationship btw folic acid + tetrahydrofolate

deficiencies cause neural tube defects, nervous system messed up cuz cant make thymines

• homocyteines accumulate bc less methionine synthase bc no THF

• homocysteine damages blood vessels, increases clotting

working hypothesis

relationship btw vitamin B12 + regeneration of tetrahydrofolate

needed to convert 5,10methylene-THF → 5-methyl-THF

• if no B12, homocysteine cant be converted to methionine + metabolic folates get trapped as 5-methyl-THF

• THF cant get regenerated

  • cant produce purines, pyrimidines

  • bones messed up

methyl trap hypothesis, no functional folate

relationship btw methyl trap hypothesis + folate levels

no functional folate bc trapped as 5-methyl-THF

steps of activated methyl cycle

• bc sulfonium unstable, methyl more reactive than tetrahydrofolate methyl

• methyl from adomet + s-adenosyl-methionine → s-adenosyl-homocysteine

• s-adenosyl-homocysteine → homocysteine + adenosine

• homocysteine + methyl from H4folate → methionine

  • via methionine synthase

  • relies on coenzyme B12!!

how activated methyl cycle plays role in alternate pathway

methyl groups from s-adenosyl methionine (adomet)

• activated derivative of methionine

• transfers carbons in most reduced state - methyl groups

relationship btw thymidylate synthase + regeneration of tetrahydrofolate

1. dUMP + 1C from N5,N10-methylene-THF → dTMP

2. during rxn, N5,N10-methylene-THF → N7,N8-dihydrofolate (oxidised)

3. dihydrofolate → tetrahydrofolate

   • by dihydrofolate reductase

   • very essential

role of methotrexate

competitive inhibitor of dihydrofolate reductase preventing making THF

• binds 100x better than other one

• used to treat cancers, arthritis, psoriasis

fluorouracil

converted to FdUMP in cell, and binds + inactivates thymidylate synthase

• cant make more TFH

• used to treat skin cancers

liver metabolic patterns

2-4% of bodyweight, metabolic hub

• portal vein = direct route from digestive organs to liver

• contains kupffer cells (liver macrophages)

• can do:

  • glucose-6-phosphate metabolism (quick fuel)

  • fatty acid metabolism (long term fuel)

  • amino acid metabolism (last resort)

  • alcohol metabolism (first bc toxic, disrupts others)

adipose tissue metabolic patterns

can do:

• triacylglycerol cycle

  • TAG → fatty acids + glycerol constantly, then turned back in liver

  • prevents fatty acid accumulating in blood stream

  • but some fatty acids in blood stream good for quick access in fight/flight

muscle metabolic patterns

contains 1% glycogen to degrade into glucose, ONLY aerobically

• but glucose never exported from muscle bc no G6P

• O2 for oxidative phosphorylation and ATP for gluconeogenesis from lactate from muscles

• Excess pyruvate in muscle converted to lactate to regenerate NAD⁺ and maintain glycolysis when CAC cant keep up

• fatty acids primary source of energy + glucose, ketone bodies, lactate

brain metabolic patterns

has low-Km hexokinase so can tightly trap + use glucose even if blood glucose levels low

• ensures continuous energy supply cuz brain important

• if low metabolic activity + glucose in brain, reduced attention slower reaction time, poor memory

• energy used to maintain Na+, K+, ATPase of nerves

kidney metabolic patterns

during starvation, contributes to gluconeogenesis!

• generates ammonia from glutamine to excrete H+

• a-ketoglutarate used

3 types of fuel reserves in healthy human adults

1. Glycogen stored in liver (and a small amount in muscle)

2. Triacylglycerols in adipose tissue

3. Tissue protein

what happens after last meal + why

after last meal:

• Blood glucose levels decline

• Liver glycogen stores are utilized (which initially aids in maintaining glucose homeostasis)

• At the end of day one:

  • Blood glucose has fallen

  • Insulin secretion slowed

  • Glucagon secretion increased (means release stored fuel!)

Metabolic priorities in starvation

Provide fuel to the brain (and red blood cells)

• first in the form of glucose

Preserve protein (which is somewhat incompatible with providing glucose)

• instead, use fatty acids + ketone bodies as fuel

general starvation induced metabolic changes in liver, adipose tissue, heart, brain

liver uses:

• amino acids → glucose (gluconeogenesis)

• fatty acids → ketone bodies (b-oxidation ketogenesis)

  • would usually use glucose for VLDL!!

adipose tissue:

• triacylglycerol → fatty acids + glycerol (UNCHANGED)

heart:

• ketone bodies → CO2 + H2O (CAC)

  • would usually use fatty acids!!

  • same products tho

brain:

• ketone bodies → CO2 + H2O (CAC)

  • would usually use glucose + glycolysis!!

  • same products tho