Lecture 17 (3-21) Flashcards
Cholesterol Biosynthesis: general early steps
- control at the enzyme level
- biosynthesis in the cytosol begins with two Claisen condensations
- first step is a thiolase reaction
- second step makes HMG-COA (3-hydroxy-3-methylglutaryl-CoA)
- third step: HMG-CoA reductase - is the rate-limiting step in cholesterol biosynthesis (note: 2 NADPH reactions)
Inhibiting Cholesterol Synthesis
Statins
- Statins are cholesterol synthesis inhibitors - why? they hit the rate-limiting step in cholesterol biosynthesis
- Lovastatin (mevinolin - ‘Mevacor’): is administered as an (inactive) lactone, blocks HMG-CoA reductase
- In the body (after oral ingestion of it), the lactone is hydrolyzed to mevinolinic acid, competitive (TSA!) inhibitor of the reductase, K1=0.6 nM!
- Mevinolinic acid is a transition-state analog of the tetrahedral intermediate formed in the HMG-CoA reductase reaction ( A TSA resembles the transition station the substrate molecule)
Other statins (anti cholesterol drugs) - HMG-CoA reductase inhibitors:
- Lipitor, Zarator, Advicor, Crestor, Lescol, Zocor (Simvastatin), Atorvastatin (Lipitor), Fluvastatin (Lescol), Pravastatin (Pravachol), Cervastatin (Baycol)
- Baycol muscle pain side effects in 1 in 10,000 people (the name for the condition thought of as Baycol muscle pain is called Rhabdomyolysis) –> Baycol was pulled from the market because of serious muscle problems
Alternative anti cholesterol approach (+ an example of it)
Bile Acid Sequestrant:
- sequester the bile acids so cholesterol can’t be absorbed (Welchol, Questran Light, Colestid)
- Problematic: absorption of other lipids/vitamins!
- Side effects: constipation, abdominal pain, bloating, vomiting, diarrhea, weight loss, flatulence
Colesevelam Hydrochloride: a bile acid sequestrant - has some GI side effects (because it throws off the process that gets FAs into system)
Atherosclerosis: what is it, what does it cause
- “Clogging…” or “hardening of the arteries”
- Causes myocardial infarction (heart attack), stroke, peripheral vascular disease
- main cause of death in NA and Europe
- infiltration of vessel walls with lipids and formation of atherosclerotic plaques
- Multifactorial: involvement of many genetic/environmental components
The problem:
- a stable plaque scan cause BLOCKAGE - Myocardial Infarction!
- unstable plaques lead to THROMBOSIS - stroke
Lipid Transport and Lipoproteins: function + types
- Lipoproteins are the carriers of the most lipids in the body
- unesterified fatty acids bound to albumin/other proteins
- phospholipids (PL), triacylglycerols (TAGs), cholesterol transported by lipoproteins
Types of lipoproteins:
- high-density lipoproteins (HDL) – smallest amount of lipid and smallest size
- low-density lipoproteins (LDL)
- intermediate-density lipoproteins (IDL)
- very low-density lipoproteins (VLDL)
- chylomicrons (lowest protein:lipid ratio but largest size) – largest amount of lipid and largest size
Properties of Major Lipoprotein Classes - origin of lipoproteins
- HDL: liver (ER)
- LDL: liver (synthesized from VLDL)
- IDL: circulation (remnants from VLDL after FA’s delivered)
- VLDL: liver (ER)
- chylomicrons: liver
Properties of Major Lipoprotein Classes - function of lipoproteins
- HDL: returns excess cholesterol back to liver
- LDL (from VLDL): main carrier of cholesterol and cholesterol esters
- IDL: remnants from VLDL after FA’s delivered
- VLDL: carry liver-SYNTHESIZED TAG to tissues
- chylomicrons (Lowest protein:lipid ratio but largest size): carry DIETARY TAG and cholesterol from gut to tissues
General Structure of Lipoprotein
- core of mobile TAGs and/or cholesterol ester
- surface is a PL monolayer where polar head groups face outward - why not a bilyer?
- —- If it had bilayer, it would have a hydrophobic shell but since this is a shuttle, you want polar heads outward to interact with solvent water (the phospholipids thus shield the hydrophobic lipids inside from the solvent water outside)
- cholesterol and protein inserted into PL layer
- Apoproteins: are the proteomic component of lipoprotein
Lipoproteins in Circulation: general description of what’s happening + roles of each individual type
lipoproteins in circulation are progressively dilapidated/degraded by lipases (specifically Lipoprotein Lipase)
- as this happens, they lose TAG and get smaller (VLDL become IDL become LDL)
- Chylomicrons’ main task is to carry dietary triglycerides from gut to peripheral tissues
- VLDLs do same for TAG’s synthesized in the liver (carry lipids from liver)
- Chylomicrons or VLDL’s anchored by LP lipase
- LP lipase activated by apoC-II
- LP lipase hydrolyzes TAGs
- free FAs taken up by cell (they are unloading fat)
- What remains? protein-rich REMNANTS!
- LDL receptor removes LDL from circulation
Lipoproteins in Circulation: LDL Receptor + associated condition
- Removes LDL from circulation (Apo B-100 critical), get them into the cells
Familial Hypercholesteremia (FH):
- genetic mutation in LDLR (LDL receptor)
- heterozygous of mutant LDLR gene -> premature CVD between 30-40 (incidence 1:500)
- homozygous: could lead to severe cardiovascular disease in childhood (pretty rare, incidence 1 in a million births)
Lipoproteins in Circulation: what happens?
In the capillaries of muscle and adipose cells:
- lipoprotein lipases hydrolyze triglycerides from lipoproteins
- lipoproteins get smaller, raising their density (correlation with exercise)
- VLDLs progressively converted to IDL and then LDL (they either return to the liver for reprocessing or are redirected to adipose tissues and adrenal glands)
Cholesterol homeostasis: what’s happening + endogenous vs. dietary part
- going through capillaries, unloading cargo (so tissues have fat-based source of energy) - particles built up, shrunk down, recycled, repeated
endogenous fat part: VLDLs
dietary fat part: chylomicrons
Hypercholesteremia: causes
- nurture (environmental) and - nature: the receptor - Familial defective apolipoprotein B-100 (mutation of apolipoprotein B that causes hyper cholesterolemia)
What happens in atherosclerosis? (early vs. later)
“Lesions” and Plaque Formation
Early lesion:
- ‘fatty streak’ is the first recognizable lesion
- observed in autopsied youths from 10-14 yo
Intermediate lesion:
- layers of macrophages and smooth muscle cells
Advanced lesion:
- fibrous plaques
- covered by dense connective tissue cap with embedded smooth muscle cells and T lymphocytes overlaying lipid core and necrotic debris
How are atherosclerotic plaques formed?
- this is NOT just a “high cholesterol” problem
- “Response to Injury” hypothesis: atherosclerotic plaques develop where vessel wall has been injured
- source of injury: not entirely clear, but one source is oxidized LDL!!
“Response to Injury” and Atherosclerosis: How is LDL oxidized? What’s the response to oxidized LDL?
How is LDL oxidized?
- reactive O2 species (ROS) released by macrophages (and other cells) at the arterial wall
- O2 radicals attack both protein and lipid components of LDL (LDL rich in polyunsaturated FA extremely susceptible!! - allylic oxidation)
Response to oxLDL:
- increased adherence of macrophages and T lymphocytes to affected vascular area
- macrophages migrate between endothelial cells and localize subendothelially
- due to cholesterol accumulation, macrophages become “foam cells” combine with T cells and smooth muscle cells to become a ‘fatty streak’
“Response to Injury” and Atherosclerosis: fatty streak
fatty streak:
- creates environment for platelet adhesion
- platelets release growth factors and cytokines, etc. (fibrous plaques)
Unsaturated and Saturated dietary fats + atherosclerosis (include specific types)
Unsaturated (the food fats):
- palmitoleic, oleic, linoleum, arachidonic, nervonic, etc.
- create enhanced potential for vessel damage –> fibrous plaque formation
Saturated (the bad fats):
- Lauric, mystic, palmitic, stearic, arachidic, etc.
- Not all are bad: Palmitic is considered a ‘bad guy’ and it stimulates cholesterol synthesis (bad) BUT stearic may have a null effect on CVD and may actually be a good guy!
Atherosclerotic plaques: % contribution from diet only ~15% (so enjoy a good juicy burger every now and again!)
Amino Acid Biosynthesis
- Plants and microorganisms can make all 20 amino acids and all other needed N metabolites
- In these organisms, glutamate is the source of N, via transamination (aminotransferase) reactions
Mammals:
- in a sense we are inferior
- we can make only 10 of the 20 aas
- the others are classed as “essential” amino acids and must be obtained in the diet
Non-essential amino acids
Alanine Asparagine Aspartate Cysteine Glutamate Glutamine Glycine Proline Serine Tyrosine
Essential amino acids (and mg)
Arginine: mg unknown Histidine: 14 Isoleucine: 19 Leucine: 43 Lysine: 38 Methionine: 19 Phenylalanine: 33 Threonine: 20 Tryptophan: 5 Valine: 24
Arg in Mammals
Arginine is considered an “essential amino acid”
- actually semi-essential (derived from the diet, endogenous synthesis, and turnover of proteins)
- dependent on: the developmental stage, health status
- preterm infants can’t synthesize arg - adults can
- surgery or other forms of trauma, sepsis and severe burns put an increased demand on the body for the synthesis of arg
His in Mammals
Histidine is considered an “essential amino acid”
- actually semi-essential
- adults produce enough from other amino acids (in liver) to support the body’s daily needs
- children obtain histidine through diet
- essential, especially during infancy, for growth and development
semi-essential amino acids
Arginine and Histidine
Amino Acids: transaminations
- Transaminations (often dependent on glutamate) are key for amino acid synthesis
- Means of transfer of N between aa and kept acids - aa1 + alpha-veto acid2 –> aa2 + alpha-keto acid1
glutamate + alpha-keep acid ——-(pyridoxal phosphate dependent aminotransferase)—> alpha-KG + alpha-Amino acid
- Aminotransferases (the enzymes that catalyze transamination reactions) exist for all amino acids except The and Lys.
- Ex: Glutamate + oxaloacetate ——-glutamate-aspartate aminotransferase——> alpha-KG + Aspartate (an amino acid)
Reminder: PLP (pyridoxal phosphate) catalyzes 7 classes of reactions involving amino acids (reactions with bonds to alpha-carbon of the aa, bonds in side chain)
Synthesis of Families of Amino Acids
- all amino acids are grouped into families according to the intermediates from which they are made
- alpha-Ketoglutarate - glutamate: Glu, Gln, Pro, Arg
- Aspartate - aspartate: Asp, Asn, Lys, Met, The, Ile
- Pyruvate - pyruvate: Ala, Val, Leu
- 3-Phosphoglycerate - 3-phosphoglycerate: Glycolic, Lys, Ser
- Aromatic - chorismate: Phe, tyr, trp
The alpha-Ketoglu Family and the Urea Cycle
- Glu, Gln, Pro, Arg, and sometimes Lys
Glutamate synthesized from:
- alpha ketoglutarate, histidine, ornithine, arginine, proline, or glutamine
- note: since biosynthesis, these are reversible
Glutamate is the key precursor to: hangover
- GABA, Gln –> brain
- Pro, HO-Pro –> collagen
- Asp, Asn –> metabolism
Note the importance of ornithine:
- precursor to Arg
- intermediate in urea cycle
- intermediate in Arg degradation
Biosynthesis of Arg gives us an N- recycling tool in the Urea cycle
- Arginine has a guanidino group
Amino Transferases (+ 2 examples)
- the most common compounds involved as a donor/acceptor pair in transamination reactions are glutamate and alpha-ketoglutarate (these participate in reactions with many different aminotrasnferases)
- some clinically-relevant serum aminotransferases: serum glutamate-oxaloacetate-aminotransferase (SGOT) (aka aspartate amino transferase or AST), Alanine Transaminase ALT
ALT + clinical application
(Alanine Transaminase) - a serum aminotransferase
- The ALT test detects liver damage but also: viral hepatitis, congestive heart failure, bile duct problems, infectious mononucleosis or myopathy
- ALT values are usually compared to the levels of alkaline phosphatase (ALP) and aspartate aminotransferase (AST), to help narrow which form of liver disease may be involved
Arg Synthesis and the Urea Cycle
- The guanidino group of Arg: N and C come from NH4+, HCO3- (Carbamoyl-P), the alpha-NH2 from Glu and Asp
- Breakdown of Arg in the urea cycle releases two N’s and one C as urea
- Important in N-excretion and N-balance (occurs in the livers of terrestrial vertebrates)
- Urea cycle is linked to TCA by fumarate
Urea Cycle - importance, location
Important for excreting excess systemic N resulting from excess aa intake (i.e. protein diet)
- consume ~100g protein per day - need to excrete 1 mol of excess N daily
Important because main form is NH3 - toxic to the central nervous system (coma-inducing)
- Encephalopathy
- Inhibits excitatory neurotransmitters
- Probably effective at the K+ pumping level
Confined to the liver (no surprise there!) and involves cytosolic and mitochondrial processes working in concert
- Liver dysfunction certain to have neurological ramifications, e.g., in chronic cirrhosis
Urea Cycle Disorders (UCDs): overview
- inborn erros of metabolism
- A genetic disorder caused by a deficiency of one of the enzymes in the urea cycle
- Nitrogen accumulates in the form of ammonia, a highly toxic substance, and is not removed from the body resulting in HYPERAMMONEMIA (ammonia reaches the brain through the blood, where it causes irreversible brain damage, coma and/or death)
- Many cases of urea cycle disorders go undiagnosed and/or infants born with the disorders die without a definitive diagnosis - exact incidence of cases is underestimated
- Possible that up to 20% of SID syndrome cases may be attributed to an undiagnosed inborn error of metabolism
- In April 2000, research experts at the Urea Cycle Consensus Conference estimated the incidence of the disorders at 1 in 10,000 births
Urea Cycle Disorders: names of specific hyperammonemias
Hyperammonemias:
- Arginosuccinic Aciduria: Arginiosuccinate Lyase Deficiency
- Hyperargininemia: Arginase Deficiency
- Citrullinemia: Arginiosuccinate Synthetase (ASS) Deficiency
- Carbamoyl Phosphate Synthetase-I (CPS-I) Deficiency
- Ornithine Aminotransferase Deficiency
- Ornithine Transcarbamylase (OTC) Deficiency
- N-Acetylglutamate Synthetase Deficiency Deficiency
Carbamoyl Phosphate Synthetase I (CPS-I) Deficiency
a hyperammonemia (Urea Cycle disorder)
- doctor found this in a woman who 10 hours after delivery of child became disoriented, agitated –> coma –> seizures
- died 3 days after delivery
- found to have CPS I deficiency
- she had been on little or no meat or dairy products
Overview of Nucleotide Metabolism
- sizable portion of the metabolic map
- the immense importance of these compounds is obvious
- —– AMP (–> ATP)
- —– GMP (–> GATP)
- —– other dNMPs (–> dNTP)
- First discussion on making ATP/GTP for use as building blocks
Nucleotide Biosynthesis
- Nearly all organisms synthesize purines and pyrimidines “de. novo” (important - thesis are the building blocks of genetic code)
- Many organisms also “salvage” purines and pyrimidines from diet and degradative pathways (ribose can be degraded to generate energy but purine and pyrimidine rings cannot)
- Nucleotide synthesis pathways are good targets for anti-cancer/antibacterial strategies*****
Biosynthesis of Purines: sources of atoms of purine ring
(the Frankenstein of Biomolecules)
John Buchanan “traced” the sources of all nine atoms of purine ring:
- N-1: aspartic acid
- N-3, N-9: glutamine
- C-4, C-5, N-7: glycine
- C-6: Co2
- C-2, C-8: THF - one carbon units
Inosine-5’-P Biosynthesis: overview
- 11 steps
- the purine ring is built on a ribose-5-P foundation
- goal: inosine-5’-P biosynthesis (inosine-5’-monophsophate/IMP) which is a purine nucleotide
- the riobose-5-P is from PPP
Inosine-5’-P Biosynthesis: Step 1
Ribose-5-P pyrophosphokinase:
- Ribose-5-P activated by Rib-5-P pyrophosphokinase (this step REGULATED)
- PRPP: is limiting substance for purine syntheiss
- But….PRPP is also a branch point - what can you deduce?
- –PRPP serves additional metabolic needs. therefore, the NEXT reaction (not this one) is actually the committed step in the pathway
(The purine ring is built on a ribose-5-P foundation)
Inosine-5’-P Biosynthesis: Step 2
GLutamine PRPP amidotransferase:
- Gln PRPP amidotransferase (KEY in regulation)
- changes C-1 configuration (alpha) to the beta position (need beta-glycosides)
- this N becomes N-9
- G- and A-nucleotides inhibit this step but at distinct guanine- and adenine-specific allosteric sites
- Note- glutamine-dependence
Gln PRPP amidotransferase
Site of action for Azaserine
- Antibiotic and anti-tumor agent
Glutamine analog
- Covalently binds the active site of Gln-dependent enzymes
- inhibitior/anti-tumor agent
- anti-fungal agent
Inosine-5’-P Biosynthesis: Step 3
Glycinamide ribonucl. synthetase:
- a. Glycine carboxyl activated by -P from ATP
- b. Amine attacks glycine carboxyl (–> Glycine carboxyl condenses with amine)
Inosine-5’-P Biosynthesis: Step 4
Glycinamide ribonucl. transformylase:
- First of two THF-dependent rxns
- Formyl group of N10-formyl-THF is transferred to free amino group of GAR (—> All atoms for 1st ring present now)
Inosine-5’-P Biosynthesis: Step 5
FGAM synthase:
- C-4 carbonyl forms a P-ester (from ATP) and active NH3 attacks C-4 to form imine
- TRANSAMINATION
- like reaction 2 - irreversibly inhibited by azaserine
Folate: clinical significance, involvement in pathway
- THF (folic acid) dependence in two steps (4 and 10) of Inosine-5’-P Biosynthesis provides susceptibility to folic acid analogs
- Folic acid may play a role in preventing cancer in selected tissues
- Diet rich in folic acid, high in methionine and low in alcohol reduce the risk of colon cancer
- Folic acid is protective against breast cancer
- Folic acid may also improve cognitive function (slow senility/Alzheimer’s)
Inosine-5’-P Biosynthesis: Step 6
Steps 6-8: Closing the first ring (carboxylation and attack by aspartate)
Step 6:
Aminoimidizole ribonucleotide synthetase
- closing the ring- similar in some ways to step 5
- ATP activates the formyl group by phosphorylation, facilitating attack by N
Inosine-5’-P Biosynthesis: Step 7
Steps 6-8: Closing the first ring (carboxylation and attack by aspartate)
Step 7:
Aminoimidizole ribonucleotide carboxylase
- Carboxylation results from CO2 addition at C-4 position
Inosine-5’-P Biosynthesis: Step 8
Steps 6-8: Closing the first ring (carboxylation and attack by aspartate)
Step 8:
Succinyliaminoimidizole-4-carboxamide synthetase
- Attack by the amino group of aspartate links this amino acid with the carboxyl group
Inosine-5’-P Biosynthesis: Step 9
Steps 9-11: loss of fumarate, another 1-C unit and the second ring closure
Step 9: Adenylosuccinate lyase
- 4 C’s of Asp removed as fumarate (impt. in muscle)
Inosine-5’-P Biosynthesis: Step 10
Steps 9-11: loss of fumarate, another 1-C unit and the second ring closure
Step 10: AICAR formylase
- Another 1-C addition catalyzed by THF (second THF-dependent reaction)
Inosine-5’-P Biosynthesis: Step 11
Steps 9-11: loss of fumarate, another 1-C unit and the second ring closure
Step 11: IMP Synthase
- Amino group attacks formyl group to close the second ring
Anaplerotic vs. Cataplerotic
Anaplerotic: make TCA intermediates
Cataplerotic: utilize TCA intermediates
Inosine-5’-P Biosynthesis: Overview + Analogs
- THF (Folic acid) dependence in two steps (4 & 10) provides susceptibility to folic acid analogs
- Analogs block purine synthesis (DNA replication, cell growth) by INHIBITING THE SYNTHESIS OF THF!
Sulfonamides: compete with PABA
Methotrexate: binds DHF reductase avidly (irreversible inhibitor)
Apoproteins (definition + examples)
the proteomic component of lipoprotein
Apo A-1: main protein in HDL, activates LCAT
Apo B-100: main protein in LDL, binds to LDL receptor (largest molecular weight)
Apo C-II: important in composition of chylomicrons and VLDL; acts ages Lipoprotein Lipase (smallest molecular weight)
Apo E: important in Chylomicrons, VLDL, and IDL, allowing the binding of these lipoproteins to the hepatocytes
Inosine-5’-P Biosynthesis: why 7 ATP?
6 steps use ATP (one each at steps 1.3.5.6.7.8) but accounting lists as 7
- 7 high energy phosphate bonds (7 ATP equivalents) are consumed because alpha-PRPP formation in reaction 1 followed by PPi release in reaction 2 represents the loss of 2 ATP equivalents
AST + clinical application
an aminotransferase
serum glutamate-oxaloacetate-aminotransferase (SGOT) (aka aspartate amino transferase or AST)
Blood level of AST may be elevated because:
- liver damage caused by: infection (viral hepatitis or mono), gallbladder disease, toxins (such as alcohol), cancer
- muscle damage caused by: muscle disease (polymyosin), progressive muscular dystrophy, injury (a fall, auto accident)
- kidney, heart, or liver injury
- heart failure
- kidney failure
- pancreases inflammation
- and others
