Biochemistry Firecracker questions

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Amino Acids

5 questions

0 The 9 essential amino acids

are histidine, isoleucine, leucine, lysine, methionine,phenylalanine, threonine, t ryptophan, and valine. These amino acids cannot be synthesized in human cells and must be obtained from the diet.

less  Only L-form (optical isomer) amino acids can be found in proteins.

 Mnemonic: “Help In Learning These Little Molecules Proves Truly Valuable" Help = Histidine In = Isoleucine Learning = Leucine These = Threonine Little = Lysine Molecules = Methionine Proves = Phenylalanine Truly = Tryptophan Valuable = Valine

The acidic amino acids are aspartic acid and glutamic acid. They are negatively charged at physiologic pH.

The basic amino acids are arginine, lysine, and histidine. Arginine is the most basic.Histidine does not have a charge at physiologic pH.

less  Found in high concentrations in proteins that need to bind strongly to negative

substrates. For instance, arginine and lysine are over-expressed in histones because the histones need to bind negatively charged DNA.

Amino acids are divided into glucogenic amino acids and ketogenic amino acids. Glucogenic amino acids can enter the CAC as either pyruvate, α-ketoglutarate, succinyl-CoA, fumarate, or oxaloacetate and used to produce glucose. The ketogenic amino acids are degraded to acetyl-CoA or acetoacetate and used to produce fatty acids or other ketone bodies. These are not mutually exclusive. Some amino acids are considered both glucogenic and ketogenic.

less  The exclusively ketogenic amino acids are leucine and lysine.

 The ketogenic and glucogenic amino acids

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Threonine is converted to glycine and acetyl-CoA via threonine dehydrogenase. However, some texts do not consider it a ketogenic amino acid.

 The exclusively glucogenic amino acids are alanine, arginine, asparagine, aspartic acid, cysteine, histidine, glutamine, glutamic acid, glycine, serine,

methionine, proline, and valine.

Pyruvate Reactions

0 Pyruvate can undergo 1 of 4 reactions:

1) Dehydrogenation by the Pyruvate Dehydrogenase Complex to yield acetyl-CoA less  Acetyl CoA enters the Citric Acid Cycle

2) Carboxylation by pyruvate carboxylase to yield oxaloacetate

less  Used in Citric Acid Cycle or Gluconeogenesis (first of 2 steps needed to

convert pyruvate back to PEP)

 Requires ATP

3) Transamination by alanine aminotransferase (ALT) to yield alanine

less  Tissues (e.g. muscle) that use amino acids for fuel generate glutamate

 Glutamate can donate its amino group to pyruvate, yielding alanine  Alanine is transported to the liver, which then regenerates pyruvate and

glutamate

 The pyruvate undergoes Gluconeogenesis and is sent out to the body  The glutamate ultimately enters the Urea Cycle → urea (nitrogen excretion)

4) Reduction by lactate dehydrogenase to yield lactate

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 Consumes NADH

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Cori Cycle

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During strenuous exercise when oxygen supply is insufficient, muscle cells must resort to anaerobic metabolism, where pyruvate is reduced by NADH to

form lactate and regenerateNAD+, instead of entering the Tricarboxylic Acid (TCA) Cycle. Lactate dehydrogenase(LDH) is the enzyme responsible for the reaction. The lactate produced in the muscle cells during anaerobic metabolism enters the bloodstream and is taken up by the liver. In the liver gluconeogenesis converts lactate into glucose. Glucose enters the bloodstream and is used by muscle cells, restarting the cycle.

In the Cori cycle lactate is produced in the muscle cells and converted to glucose in the liver, so the muscle cells can make more lactate. Glycolysis and anaerobic metabolism in themuscle cells generate 2 ATP per glucose; gluconeogenesis in the liver consumes 6 ATPto generate one glucose from two lactate. Overall, 4 net ATP are consumed for each round of the Cori cycle; therefore, there is a metabolic shift to the liver.

Red blood cells, which lack mitochondria, produce lactate and hence participate in the Cori cycle.

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The Pyruvate Dehydrogenase Complex (PDC): converts pyruvate into acetyl-CoA through several reactions, linking glycolysis (cytoplasm) and the citric acid cycle (mitochondria)

less  PDC is located in the mitochondrial matrix

 The transport of pyruvate into mitochondria consumes energy, lowering the total ATPproduction of aerobic glucose metabolism

 The complex has 3 enzymes: E1 (pyruvate dehydrogenase), E2, and E3  Reaction: pyruvate + NAD+ + CoA → acetyl-CoA + CO

2 + NADH

E1 requires, as a cofactor, thiamine pyrophosphate (TPP), a derivative of vitamin B1

less  The rate limiting step of the reaction

E2 requires, as cofactors, CoA and lipoic acid

less  It is this step that produces acetyl-CoA (hence the need for CoA in this step)

E3 requires, as cofactors, FAD (vitamin B2) and NAD+ (vitamin B3)

less  FAD oxidizes a lipoic acid intermediate back to lipoic acid so it can participate in more reactions → in the process, FAD is reduced to FADH2 → FADH2 is then used to reduce NAD+ to NADH

The PDH complex is regulated directly through phosphorylation

less  PDH kinase and PDH phosphatase are part of the PDH complex and act on

E1.

Phosphorylation through PDH kinase inhibits E1, while dephosphorylation through PDHphosphatase activates E1

 PDH kinase is activated (which leads to inactivation of E1) by ATP, acetyl-CoA, andNADH

PDH kinase is inhibited (which leads to activation of E1) by pyruvate PDC deficiency has two typical presentations:

1. Metabolic (lactic acidosis as pyruvate is shunted to lactate)

2. Neurological (hypotonia, poor feeding, lethargy, seizures, mental retardation) less  Most common form is caused by mutations in the X-linked E1 gene

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Even though the E1 mutation is X-linked, it still affects females due to critical role of the enzyme in the nervous system → considered X-linked dominant

 Key feature: gray matter degeneration with brainstem necrosis and capillary proliferation

 Treatment: very few forms respond to cofactor supplementation with thiamine  Ketogenic diets (high fat, low carbohydrate, adequate protein) have minimal

success

Citric Acid Cycle

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Citric Acid Cycle: central catabolic pathway used to generate energy through the oxidization of acetate (derived from carbohydrates, fats, and proteins) into CO2 and H2O

For each turn, the cycle produces 1 GTP, 3 NADH, 1 FADH2, and 2 CO2

less  1 NADH → 3 ATP equivalents

1 FADH2 → 2 ATP equivalents

However, because of the energy expenditure required to shuttle NADH and FADH to theETC, each turn through the citric acid cycle yields:

3 NADH x 2.5 → 7.5 ATP equivalent 1 FADH2 x 1.5 → 1.5 ATP equivalent 1 GTP → 1 ATP equivalent

For a total of 12 potential and 10 actual ATPs

The citric acid cycle (tricarboxylic acid cycle or Krebs cycle) takes place in the mitochondrial matrix

Citrate synthase catalyzes the transfer of a 2-carbon acetyl group from acetyl-CoA tooxaloacetate, forming the 6-carbon molecule citrate

less  Citrate synthase is inhibited by ATP, NADH and succinyl CoA and stimulated

by insulin

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Aconitase catalyzes the isomerization of citrate into isocitrate

less  Fluoroacetate (a metabolic poison) inhibits the enzyme aconitase

Isocitrate dehydrogenase catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate

less  NAD+ → NADH, 1st molecule of CO

2 is released

 Key regulatory step that is stimulated by ADP (low energy state) and inhibited by ATP andNADH (high energy state)

The α-ketoglutarate dehydrogenase complex converts α-ketoglutarate to succinyl-CoA

less  NAD+ → NADH, 2nd molecule of CO

2 is released

 Regulatory step, regenerates a 4-carbon chain (CoA excluded) and requires many coenzymes, including vitamins B1, B2, B3, CoA, and lipoic acid

Note: the same cofactors are required in the pyruvate dehydrogenase complex.  α-ketoglutarate dehydrogenase is inhibited by NADH, succinyl

CoA, ATP and GTP

Succinyl-CoA synthetase converts succinyl-CoA to succinate and CoA

less  Substrate level phosphorylation: GDP + Pi → GTP

The succinate dehydrogenase complex catalyzes oxidation of succinate to fumarate less

 FAD → FADH2

Mitochondrial fumarase converts fumarate to malate

Malate dehydrogenase oxidizes malate to oxaloacetate, and the cycle can begin anew

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 NAD+ → NADH

Electron Transport Chain

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Electron transport chain: uses NADH and FADH2 electrons (from glycolysis, pyruvate dehydrogenase complex, and the citric acid cycle) to form a proton gradient, coupled to oxidative phosphorylation, that drives the production of ATP

less  The ETC (electron transport chain) is composed of 5 multi-enzyme

complexes, numbered I-V, that accept and donate electrons while molecular oxygen, O2, is the final electron acceptor

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 Mobile electron carriers, such as cytochrome c and coenzyme Q, shuttle electrons between various enzyme complexes of the ETC

 Primary NADH electron transport system: malate-aspartate shuttle, which transportsNADH electrons to complex 1 in the mitochondria.

 Less commonly used NADH electron transport system: glycerol-3-phosphate shuttle

 Theoretically: 1 NADH yields 3 ATP and 1 FADH2 yields 2 ATP (because FADH2 electrons are transferred to complex II, a lower energy level than NADH) However, since NADH from glycolysis needs to be transported into the mitochondria and the mitochondrial membrane "leaks" protons, the actual yields are smaller  As electrons flow through the ETC, protons (H+) are pumped into the

mitochondrial inter-membrane space → this creates an electrochemical proton gradient

 ATP Synthase (Complex V): uses the electrochemical proton gradient created by theETC to pump protons (H+) back into the mitochondrial matrix to produce ATP from ADPand Pi

Toxins that disrupt any component of the ETC disrupt the aerobic production of ATP → tissues that depend highly on aerobic respiration, such as the CNS and the heart are particularly affected

less  Amobarbital (known as amytal) and rotenONE bind to NADH dehydrogenase

(complex 1) → directly inhibit electron transport

Antimycin A binds to cytochrome c reductase (complex III) → directly inhibits electron transport

 Carbon monoxide and Cyanide bind to Cytochrome C oxidase (complex IV) → directly inhibit electron transport

 Oligomycin (a macrolide) inhibits ATP synthase (complex V) by blocking its proton channel

 2,4-Dinitrophenol and ↑ doses of aspirin increase the permeability of the inner mitochondrial membrane → ↓ proton gradient and ↑ oxygen consumption → heat generated instead of ATP (explains the fever generated following toxic doses of aspirin)

 Thermogenin in brown fat is an uncoupling agent that disrupts the proton gradient → used to generate heat in animals

HMP Shunt (Pentose phosphate pathway)

4 questions

0

HMP Shunt: a 2 phase pathway consisting of an oxidative (irreversible) phase andnonoxidative (reversible) phase that uses available glucose-6-phosphate to mainly produce NADPH and ribose-5-phosphate

less  Both phases occur in cytosol

 ATP is not used or produced!

 Key reactions:

1) Glucose-6-Phospate → Ribulose-5-Phospate + 2 NADPH *Enzyme: Glucose-6-Phosphate Dehydrogenase

2) Ribulose-5-Phosphate → → Ribose-5-Phosphate + G3P + F6P *Enzyme: Transketolase

Oxidative phase: key enzyme is G6P dehydrogenase (G6PD), the rate-limiting enzyme; all steps of the oxidative phase are irreversible and are used to

generate NADPH for reductive biosynthetic pathways

less  NADPH is used to reduce glutathione, a coenzyme for glutathione

peroxidase which prevents oxidative damage by converting H2O2 → H2O. This is especially important in RBCs

 Increased in tissues that consume NADPH in reductive pathways like adipose tissue for fatty acid synthesis, gonads and adrenal cortex for steroid synthesis, liver for fatty acid and cholesterol synthesis, and glutathione reduction inside RBCs Nonoxidative phase: key enzyme is transketolase (thiamine-dependent); all steps arereversible and are used to convert sugars to produce ribose-5-phosphate and intermediates for glycolysis and gluconeogensis

less  Pentose sugars like ribose-5-phosphate are used for nucleotide synthesis  Fructose-6-phosphate and glyceraldehyde 3-phosphate (products of the

non-oxidative phase) are used as substrates for glycolysis in fed state, and intermediates in gluconeogenesis in the fasting state

G6PD deficiency: hemolytic anemia when RBCs are exposed to oxidative stress because of inadequate NADPH production leading to less anti-oxidant activity of glutathione

less  Causes of oxidizing stress: infections, fava beans, drugs (e.g. sulfonamides,

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 Transmitted in X-linked recessive fashion with a predominance in Asia, the Mediterranean, and Africa (disease provides protection against Plasmodium falciparum malaria)

 On a peripheral smear look for Heinz bodies (inclusions in RBCs composed of denaturedHemoglobin) and degmacytes (bite cells) (result of splenic

macrophages removing Heinz bodies)

Mono/Disaccharide Metabolic Disorders

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Hereditary fructose intolerance: autosomal recessive deficiency of aldolase B, which cleaves fructose 1-phosphate to 3-carbon molecules.

less  A deficiency in aldolase B leads to accumulation of phosphorylated fructose

→ available phosphate levels drop → gluconeogenesis is blocked  Symptoms: hypoglycemia, vomiting, jaundice, and cirrhosis

 Patients usually asymptomatic until challenged, in infancy, with fructose  Treatment: avoid intake of fructose or sucrose (combination of glucose and

fructose)

Essential fructosuria: autosomal recessive, benign condition resulting from defect in hepatic fructokinase

less  Fructose can not be phosphorylated, so it is unable to be sequestered in the

cell → elevated serum fructose levels → fructosuria

Classic galactosemia: autosomal recessive deficiency in GALT (galactose-1-phosphate uridyl transferase), which converts galactose-1-(galactose-1-phosphate to glucose-1-phosphate

less  Absence of GALT leads to galactose-1-phosphate accumulation → toxic  All states mandate neonatal screening because lactose (i.e. milk) is

metabolized to glucose and galactose

 Symptoms: poor growth, hepatic dysfunction (jaundice, coagulopathy, hepatomegaly), ascites, cataracts, mental retardation

These infants also have an ↑ risk for E. coli septicemia.  Treatment: galactose-free diet

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Galactokinase deficiency: autosomal recessive deficiency in galactokinase, which phosphorylates galactose to make galactose-1-phosphate

less  Accumulation of galactose → galactosemia → galactosuria

 Galactosemia → cataracts because the lens of the eye contains aldose reductase, which converts galactose to galactitol, an osmotically active alcohol Lactase deficiency: age-related or hereditary lactose intolerance due to ↓

expression of lactase (a brush-border enzyme) or transient ↓ expression following gastroenteritis

less  Symptoms: osmotic diarrhea, bloating/cramps

 Treatment: avoid lactose

Sorbitol accumulation: high blood levels of glucose (or fructose or galactose) lead to osmotic damage from sorbitol accumulation in tissues that lack sorbitol

dehydrogenase → cataracts, diabetic retinopathy, and peripheral neuropathy

less  Liver, ovaries, and seminal vesicles have both aldose reductase and sorbitol

dehydrogenase (thus, there is no sorbitol accumulation)

Glucose → sorbitol (via aldose reductase) → fructose via (sorbitol dehydrogenase)

 Schwann cells, lens, retina, and kidneys only have aldose reductase (thus, there is sorbitol accumulation in hyperglycemic states)

Phenylalanine & Tyrosine Metabolism

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Phenylalanine hydroxylase (PAH): enzyme that converts Phenylalanine to Tyrosine less  In this reaction, tetrahydrobiopterin (BH4), the required cofactor, is converted

to dihydrobiopterin (BH2)

 BH2 is converted back to BH4 via the enzyme dihydrobiopterin reductase  Tyrosine is a precursor for many catecholamines, neurotransmitters, melanin

and thyroid hormones

Phenylketonuria (PKU): autosomal recessive defects in the enzyme phenylalanine hydroxylase (PAH)

less  Phenylalanine accumulates and leads to the following symptoms:

- neurologic defects (e.g. seizures and mental retardation) - albinism (tyrosine required for melanin synthesis)

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- "musty odor" to their sweat & urine (due to accumulated phenylalanine conversion to phenylketones)

 Screened for on the 2nd or 3rd day of life due to presence of maternal enzyme at birth

 Treatment: restrict phenylalanine and aspartame (contains phenylalanine) in diet and ↑ tyrosine intake (becomes an essential amino acid)

Maternal PKU: lack of proper dietary treatment in a pregnant woman with PKU → infant born with microcephaly, congenital heart defects, mental and growth

retardation

Malignant PKU: autosomal recessive defects in the enzyme dihydrobiopterin reductase (called malignant because restricting phenylalanine does not correct neurological problems)

 Note: in malignant PKU, BH2 is not converted back to BH4 because of a defect in dihydrobiopterin reductase so DOPA needs to be supplemented in these patients Tyrosine hydroxylase: enzyme that converts Tyrosine to Di-hydrOxy-PhenylAlanine (orDOPA)

less  (BH4) is a necessary co-factor for the enzyme tyrosine hydroxylase. (BH4) is

also a co-factor for phenylalanine hydroxylase.

Tyrosinase: similar to tyrosine hydroxylase in that it converts Tyrosine to DOPA, but this enzyme has further catalytic activity that results in the production of melanin from DOPA

less  Autosomal recessive defects in tyrosinase or albinism: absence of melanin

in hair (white hair), eyes (photophobia), and skin (increased risk of UV related skin cancer

Homogentisic acid dioxygenase (HGD): enzyme that is part of the degradative pathway of tyrosine into fumarate

less  The catabolic process involves homogentisate (or alkapton) as an

intermediate

 Congenital deficiency of HGD (or alkaptonuria), autosomal recessive disease with the following symptoms:

- ↑ homogentisate → excreted in urine (if the urine is left standing it will turn black) - homogentisate also polymerizes and deposits in joints → joint arthritis, ankylosis, and arthralgias (toxic to cartilage)

- dark connective tissue (called ochronosis) - brown hyper-pigmented sclera

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Branched-chain Ketoaciduria (Maple Syrup

Urine Disease)

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Branched chain ketoaciduria (maple syrup urine disease): autosomal

recessive defect in the branched-chain α-ketoacid dehydrogenase complex (BCKD) less  The BCKD complex catalyzes the breakdown of Isoleucine, Leucine,

and Valine

Mnemonic: I Love Vermont maple syrup from trees with branches

 Defective BCKD → accumulation of branched chain amino acids in the blood and the brain → irreversible neurological damage

 Symptoms typically present in the first few days of life (days 4-7) and include poor feeding, vomiting, poor weight gain, lethargy, and maple syrup odor to the urine  Isoleucine: characteristic maple syrup odor of the urine

 Leucine: readily crosses the blood-brain barrier and is responsible for the neurological symptoms

 Treatment: restrict amino acid intake, and a small number of patients respond to thiamine (vitamin B1) supplementation

Lipoprotein Complexes and

Apolipoproteins

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Lipoprotein complexes are composed of cholesterol, TGs (triglycerides), and phospholipids and apolipoproteins.

less  Lipoprotein complexes include: chylomicrons, VLDL, IDL, LDL, and HDL

Apolipoproteins are proteins that bind to lipids; they have various functions:

less  ApoA-I activates LCAT

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 ApoB-100 is the sole protein component of LDL

 ApoB-48 lacks the LDL-receptor binding sequence that ApoB-100 has. It is a component of chylomicrons.

 ApoC-II activates lipoprotein lipase (LPL) in capillaries  ApoE mediates chylomicron and IDL uptake in the liver.

Glucose Transport

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All GLUT transporters work via facilitated diffusion GLUT1: most cell types including RBCs and brain

GLUT2 (bidirectional): pancreatic β islet cells, liver, renal tubular cells, small intestine less  Low affinity and high capacity isoform – liver needs to be

glycogen/glucose reservoir, but shouldn’t compete with other tissues

This should look similar to glucokinase (low affinity or ↑ Km and high capacity or ↑ Vmax) because it has a similar tissue distribution

GLUT3: neurons, testes, and the placenta

GLUT4: adipose tissue and striated muscle (skeletal and cardiac)

less  Insulin regulates insertion of GLUT4 transporters into cell membrane in

response to high glucose levels

Ketones

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Acetoacetate, β-hydroxybutyrate, and acetone are the ketones produced during ketogenesis: Produced by liver for use in the brain and heart.

less  Most other tissues can use fatty acids, but brain cannot. The liver lacks the

enzymes to use ketones

During hypoglycemia, fatty acids are sent to the liver for oxidation → ↑ acetyl-CoA levels.

less  The rate limiting enzyme in the formation of ketones is HMG-CoA synthase.

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Ketone utilization: If the ketone is acetoacetate, this is converted (via multiple steps) to 2 acetyl-CoA molecules that enter the TCA cycle

less  If the ketone is β-hydroxybutyrate, it is converted back to acetoacetate, then

→ → 2 acetyl-CoA

Ketones are excreted in urine. Acetone, from spontaneous decarboxylation of acetoacetate, causes the "fruity odor" detected on breath during ketoacidosis Diabetic ketoacidosis: ↓ insulin (mostly in Type I diabetes) leads to ↑ ketone production because cells are unable to utilize serum glucose without insulin (↓ glucose in cells → oxaloacetate is shunted into gluconeogenesis → stops the TCA cycle → acetyl CoA is shunted into ketogenesis)

Methylmalonic and Propionic Acidemia

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Methylmalonic acidemia and Propionic acidemia: autosomal recessive disorders of enzymes in the pathway that converts propionyl CoA to succinyl CoA (process that produces energy or glucose from odd-chain fatty acids or certain amino acids)

less  Pathway: propionyl CoA → methylmalonyl CoA → succinyl CoA

 Propionyl CoA carboxylase: enzyme that catalyzes the conversion of proprionyl CoA to methylmalonyl CoA (deficiency of this enzyme leads to propionic acidemia)

 Methylmalonyl CoA mutase: enzyme that catalyzes the conversion of methylmalonyl CoA to succinyl CoA, requiring vitamin B12 as a cofactor (deficiency of this enzyme leads to methylmalonic acidemia)

 Symptoms of both include: ketosis, metabolic acidosis, vomiting, lethargy, poor feeding, neutropenia, and developmental/neurological complications

 Propionic acidemia → ↑ levels of propionic acid in the blood

Methylmalonic acidemia → ↑ levels of propionic acid and ↑ levels of methylmalonic acid in the blood

Need to rule out vitamin B12 deficiency with methylmalonic acidemia because some neurologic symptoms are reversible

Treatment for both: low-protein diet (specifically ↓ intake of methionine, valine, threonine, isoleucine, and odd-chain fatty acids because they are all broken down

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into propionyl CoA) and carnitine supplementation (improves β-oxidation of fatty acids)

Hemoglobin (Hb)

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Tetramer (4 subunits); each subunit polypeptide has a heme molecule at its center and each heme molecule can carry 1 oxygen molecule

less

 Hemoglobin A (adult): α2β2

 Hemoglobin A2 (adult): α2δ2

 Hemoglobin F (fetal): α2γ2 – elevated in sickle-cell disease patients

Hemoglobin’s oxygen dissociation curve is sigmoidal: the tetramer flips between 2 conformations

less  Deoxy or T (Taut) form: low O2 affinity

 Oxy or R (Relaxed) form: high (↑ 300x) O2 affinity

 When 2 O2 molecules are bound to the T form, conformation switches to R and all 4 sites can be filled

 The T to R shift occurs under conditions of high oxygen tension (i.e. the lungs) and the R to T shift occurs under conditions of low oxygen tension. Lung: High O2 → oxygenated Hb.

Tissues: Low O2 → deoxygenated Hb (Bohr effect: ↑ CO2 and/or H+ concentration stabilizes deoxygenated conformation).

4 factors cause O2 dissociation (T form favored):

less  1) ↓ pH: relative acidic environment, like peripheral tissues

 2) CO2: produced by cellular metabolism

 3) 2,3-DPG (diphosphoglycerate, the same as bisphosphoglycerate): stabilizes the T conformation, produced by glycolysis

 4) ↑ temperature

 These factors shift the O2 dissociation curve to the right – a higher O2 pressure is needed to maintain the same level of hemoglobin saturation

Myoglobin has a similar structure/sequence, but is a monomer → doesn’t exhibit cooperative binding

CO2 transport: CO2 is converted to H2CO3 by carbonic anhydrase

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 H2CO3 (carbonic acid) dissociates to bicarbonate and a proton; the H+ binds to hemoglobin and thus has no effect on serum pH

 Allosteric inhibition: CO2 also binds at the hemoglobin chain N terminus, favoring the deoxy Hb form

Carbon monoxide: CO is a competitive inhibitor with 200x affinity for heme compared to O2

less  Carboxyhemoglobin is bright red and poisoned patients are commonly

described as having a cherry-red appearance to their skin Iron in Hb is usually in the Fe2+ (ferrous), reduced state

less  Methemoglobinemia: oxidation to the Fe3+ (ferric) state leads to decreased

affinity of O2at these heme sites; however, at other non-oxidized heme sites, there is a compensatory increase in affinity → leading to a left shift of the

oxygen-dissociation curve

 Normally, oxidation is prevented via a reductive enzyme pathway (HMP shunt) in RBCs

 Drugs that cause

methemoglobinemia: Metoclopramide, Procaine, Nitrites, Antimalarials,Sulfonamides , Dapsone.

Can be easily remembered with mnemonic: A Methemoglobinemic Patient is Not AlwaysSomething Deadly.

 Treatment: methylene blue

Cyanide poisoning: CN- preferentially binds to Fe3+ and inactivates cytochrome c oxidasein the electron transport chain → stops cellular respiration

less  Nitrites can be used to convert Hb to methemoglobin → methemoglobin then

binds the CN-→ use sodium thiosulfate to chelate this CN- and yield thiocyanate → renally excreted

 Methemoglobinemia decreases the patient’s O2 carrying capacity, but methemoglobinemia can be managed whereas arrested cellular respiration is irreversible

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SAM (S-Adenosyl Methionine)

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SAM (S-Adenosyl Methionine): the primary methyl donor of the body

less  Helps methylate DNA

 After donating its methyl group, SAM is hydrolyzed to homocysteine and adenosine; regeneration of methionine from homocysteine requires folate and vitamin B12

Fatty Acid Oxidation

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In the cytosol, long chain (> 14 C) free fatty acids are converted to fatty acyl-CoA by fatty acyl CoA synthetase. This step activates the fatty acid for transport into the mitochondria.

Because the inner mitochondrial membrane is impermeable to CoA, the carnitine shuttle system is required to transport the fatty acyl CoA into the mitochondrial matrix.

less

 Step 1:

Enzyme: CAT-I (carnitine acyl transferase I) on the outer mitochondrial membrane Reaction: Fatty acyl-CoA + carnitine → fatty acyl carnitine + free CoA

CoA remains in the cytosol, and fatty acyl carnitine can now pass through the inner mitochondrial membrane.

 Step 2:

Enzyme: CAT-II on the inner surface of the inner mitochondrial membrane

Reaction: Fatty acyl carnitine + CoA (already in the mitochondrial matrix) → fatty acyl CoA + free carnitine

Fatty acyl CoA stays in the mitochondrial matrix for further metabolism, and carnitine leaves the matrix to be used again in the shuttle.

 Carnitine deficiency → decreased ability to utilize long chain fatty acids as a fuel source. Can be due to environmental (e.g. malnutrition) or genetic factors (e.g. CAT-I deficiency).

Symptoms: Muscle aches and fatigue following exercise, ↑ free fatty acid levels in the blood, hypoketotic hypoglycemia.

Treatment: Diet high in carbohydrates and medium and short chain fatty acids, low in long chain fatty acids.

 Malonyl-CoA, an intermediate in fatty acid biosynthesis, inhibits this shuttle system to prevent newly synthesized fatty acids from entering the degradation pathway, and thus prevent a futile synthesis-degradation cycle

Medium and short chain fatty acids directly enter the mitochondrial matrix without need for a special transport.

less  In the mitochondrial matrix, fatty acyl-CoA synthetase activates short/medium

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 MCADD (medium-chain acyl-CoA dehydrogenase deficiency): MCAD is a enzyme required for complete oxidation of medium length fatty acids. Deficiency → inability to oxidize fatty acids with <12 carbons.

Presents with symptoms of hypoglycemia.

Treatment: Avoid prolonged fasting, ↑ carbohydrate and protein intake, ↓ fat intake. Inside the mitochondria, fatty acyl-CoA with an even number of carbons undergo successive rounds of β oxidation, yielding acetyl-CoA, and NADH & FADH2.

less  Acetyl-CoA enters the citric acid cycle.

 NADH and FADH~2~ are used in the electron transport chain. Oxidation of fatty acids containing an odd number of carbons → Acetyl-CoA and Propionyl-Acetyl-CoA

less  Because Propionyl-CoA → Succinyl-CoA, it is the only part of fatty acids that

isgluconeogenic.

Ketogenesis occurs when there is a high rate of fatty acid oxidation forming Acetyl-CoA

less  When the liver is overloaded with Acetyl-CoA → ketone bodies form

 The 2 main ketone bodies are acetoacetate and β-hydroxybutyrate  Acetoacetate undergoes spontaneous decarboxylation to form acetone  Ketones are generally used in 2 ways:

1) Extrahepatic tissues can convert ketone bodies → Acetyl-CoA

2) Because ketone bodies are volatile, they are readily exhaled by the lungs (Note: This is why diabetics in DKA have “fruity” smelling breath)

Regulation of Glycolysis

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Regulation points indicate places where intermediates can be shunted in and out of glycolysis. Examples:

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pathway after the 1st regulated reaction.

2. Shunting out of glycolysis: DHAP can be turned into glycerol-3-phosphate instead ofGADP. Glycerol-3-phosphate is then shunted into triglycerides synthesis. There are 3 key regulated glycolytic enzymes: Hexokinase/glucokinase, PFK-1 and pyruvate kinase

Hexokinase: Inhibited by G6P and not induced by insulin Glucokinase: Not inhibited by G6P but is induced by insulin Remember glucokinase is induced by insulin.

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 Hexokinase: High affinity (↓ Km), low capacity (↓ Vmax)

 Glucokinase: Low affinity (↑ Km), high capacity (↑ Vmax) GLUcokinase is a GLUtton with an insatiable Vmax

PFK-1 activity is ↑ by AMP and F2,6BP and ↓ by acetyl-CoA, citrate, and ATP

less  PFK-2/FBPase-2 (phosphofructokinase-2/Fructose bisphosphatase-2) is a

bifunctional enzyme:

1. PFK-2 synthesizes F2,6BP (fructose-2,6-bisphosphate) from F6P, the same substrate from which F1,6BP is synthesized

2. FBPase-2 reverses the reaction, by dephosphorylating F2,6BP to form F6P  F2,6BP is the most potent activator of PFK-1

 High glucose levels ↑ insulin → dephosphorylation of PFK-2/FBPase-2 → activatesPFK-2 activity while simultaneously inhibiting FBPase-2 activity → F6P converted to F2,6BP → stimulates glycolysis by activating PFK-1

 Low glucose levels ↑ glucagon → ↑ cAMP and pKA activity → phosphorylation of PFK-2/FBPase-2 → activates FBPase-2 activity while

simultaneously inhibiting PFK-2 activity → F2,6BP converted to F6P → decrease in PFK-1 activity inhibits glycolysis

Pyruvate kinase activity is ↑ by F1,6BP and PEP, while allosterically inhibited by ATP and alanine

less  Low glucose leads to ↑ glucagon levels → ↑ cAMP and pKA activity →

causing phosphorylation and inactivation of pyruvate kinase → allows PEP to enter gluconeogenesis

(21)

(22)

Glycogen Storage Diseases

0

Glycogenoses (glycogen storage diseases): autosomal recessive enzyme defects that result in either decreased glycogen breakdown (eg, Pompe’s) or

increased glycogen synthesis (eg, von Gierke’s) → accumulation of glycogen within the cytoplasm (except Pompe’s, where glycogen accumulates in lysosomes) of cells in one or more tissues → organ dysfunction

less  Mnemonic: If you have Very Poor CArbohydrate Metabolism, don’t eat

that Hershey’s bar! Type 1: Von Gierke’s Type 2: Pompe’s Type 3: Cori’s Type 4: Andersen’s Type 5: McArdle’s Type 6: Hers’

Type I (von Gierke’s): deficient glucose-6-phosphatase (liver, kidney) → defective glycogenolysis and gluconeogenesis → severe fasting hypoglycemia (seizures, hypoxic brain damage)

less  Normal glycogen structure

 Hepatorenomegaly – glycogen accumulates in liver and kidney because excess G-6-P stimulates glycogen synthesis and inhibits glycogenolysis

 Hyperlipidemia → skin xanthomas and ↑ VLDL

 Hyperuricemia – ↓ free phosphate due to G6-Pase defect →

↑ AMP → AMP degraded to uric acid → ↑ uric acid → predisposes to gout  Fasting lactic acidosis – ↑ lactate → ↓ uric acid excretion by kidneys  Ingestion of galactose or fructose → no increase in blood glucose

(23)

 Administration of glucagon, epinephrine, or other gluconeogenic stimulus → no increase in blood glucose

Type II (Pompe’s): defective lysosomal α-1,4-glucosidase → enzyme is

responsible for digesting glycogen → glycogen deposits accumulate in lysosomes less  Lysosomal α-1,4-glucosidase is responsible for only ~3% of glycogenolysis,

so defects don’t cause hypoglycemia

 Organs most affected are those that store glycogen (liver, heart, skeletal muscle)

 Left ventricular hypertrophy leads to outflow tract obstruction and cardiac failure

 Mortality in infantile form due to cardiac failure from massive cardiomegaly → death before age 2

 Mnemonic: Pompe’s trashes the Pump (heart)

Type III (Cori’s): defective α-1,6-glucosidase (glycogen debrancher enzyme) → fasting hypoglycemia → milder than Type I (von Gierke’s), normal blood lactate levels

less  Glycogen molecules have shorter outer branches → single glucose residue  Glycogenolysis is defective but gluconeogenesis is still functional.

 Presents with hepatomegaly in infancy

Type IV (Andersen’s): defective α-4,6-glucosidase (glycogen branching enzyme)

less  Inability to form branches → accumulation of long, insoluble glycogen chains

→ hepatosplenomegaly and cirrhosis

 Causes infantile cirrhosis failure to thrive and hypotonia → usually fatal Type V (McArdle’s): defective skeletal muscle glycogen phosphorylase → unable to break down glycogen → ↑ glycogen in muscle → muscle

cramps/weakness with exercise → can lead to myoglobinuria

less  Still form normal glycogen molecules

 McArdle’s = Muscle phosphorylase

Type VI (Hers’): deficient Hepatic glycogen phosphorylase → gluconeogenesis but no glycogenolysis → fasting hypoglycemia (mild) and hepatomegaly/cirrhosis

less  Hers’ = Hepatic phosphorylase

 As with Type V (McArdle’s), glycogen structure is normal

 Early childhood presentation of hepatomegaly and growth retardation → hepatomegaly may improve with ↑ age

Familial Dyslipidemias/Hyperlipidemias

4 questions

0

In 1967, Dr. Donald S. Fredrickson developed a classification of lipoprotein

abnormalities, depending on the pattern of lipoprotein electrophoresis. Collectively, they are called dyslipoproteinemias, but most have a more descriptive, colloquial name. Lipid Digestion & Metabolism and the role of each Lipoprotein Complexes and Apolipoproteins are not included in this topic.

Type I dyslipoproteinemia, or more commonly hyperchylomicronemia, is

characterized by a deficiency in lipoprotein lipase (LPL) or a mutation in ApoC-II. The inheritance pattern of hyperchylomicronemia is autosomal recessive.

less  Due to defective LPL or ApoC-II, the chylomicrons cannot be cleared from the

blood. Lab findings include increased serum chylomicrons, triglycerides, and cholesterol.

 The clinical findings include chylomicron-induced

acute pancreatitis,hepatosplenomegaly, and eruptive/pruritic xanthomas.  There is no increased risk of atherosclerosis.

Type IIa dyslipoproteinemia, or more commonly familial hypercholesterolemia, is characterized by an absence of or a decrease in competent LDL receptors. The inheritance pattern is autosomal dominant.

less  Due to defective LDL receptor, LDL cannot be cleared from the blood. Lab

findings include increased serum LDL and cholesterol leading to increased risk of atherosclerosis and coronary artery disease.

 Patients develop tendon xanthomas, classically on the Achilles’ heel and, less commonly, elbow.

 Patients develop corneal arcs (a.k.a., arcus senilis, arcus senilis corneae), which is gray or white arcs of lipid deposits visible at the edge of the cornea. Note: This is also seen is other dyslipidemias.

 Treatments:

Cholestyramine and other drugs that ↑ bile salt synthesis from cholesterol → ↓ cholesterol concentration within hepatocytes → ↑ LDL receptor expression → ↑ cholesterol removal from circulation.

Statins ↓ de novo synthesis of cholesterol → increases LDL receptor expression  Prevalence: heterozygotes = 1/500 (cholesterol > 300 mg/dL); homozygotes =

(25)

 Heterozygotes (1 in 500) have total serum cholesterol around 300 mg/dL. Homozygotes (very rare) have total serum cholesterol 700 mg/dL or greater and have poor prognosis due to myocardial infarction before age 20.

Type IIb dyslipoproteinemia, or more commonly familial combined hyperlipidemia, is characterized by decreased LDL receptor and increased ApoB. The mechanism is not fully elucidated. The inheritance pattern is autosomal dominant.

less  Due to decreased LDL receptor and increased ApoB, the characteristics lab

findings areincreased serum LDL, VLDL, and triglycerides (<1000 mg/dL).  Familial combined hyperlipidemia affects 1% of the population.

Type III dyslipoproteinemia, or more commonly, dysbetalipoproteinemia, is

characterized by a mutation in ApoE. The name dysbetalipoproteinemia comes from the broadening of the beta band, which contains increased

chylomicrons/VLDL/IDL/LDL, on lipoprotein electrophoresis.

less  Due to defective ApoE, chylomicrons and and IDL cannot be cleared from the blood. The lab findings include increased serum IDL, cholesterol, and triglycerides and decreased serum HDL.

 Presents with tuberoeruptive xanthomas and peripheral vascular disease. Tuperoeruptive xanthomas, as the name suggests, are large grapelike nodules that appear inflamed and coalesce.

 Responsive to dietary changes and cholesterol lowering drugs (statins). Type IV dyslipoproteinemia, or more commonly hypertriglyceridemia, is

characterized byincreased VLDL production. The inheritance pattern is autosomal dominant.

less  Due to hepatic overproduction of VLDL, the characteristic lab findings

are increased serum VLDL and triglycerides.  Patients present with pancreatitis.

Type V dyslipoproteinemia is characterized

by increased VLDL production anddecrease lipoprotein lipase production. less  The lab findings are increased serum chylomicrons and VLDL.

The high yield dyslipoproteinemias are hyperchylomicronemia (Type I dyslipoproteinemia),hypercholesterolemia (Type IIa dyslipoproteinemia), and hypertriglyceridemia (Type IV dyslipoproteinemia).

Abetalipoproteinemia (also known as Bassen-Kornzweig syndrome) is

characterized by microsomal triglyceride transfer protein (MTTP) deficiency. MTTP is essential for synthesis and secretion of beta-type apolipoproteins (e.g., ApoB-48 and ApoB-100). The inheritance pattern is autosomal recessive.

(26)

 Due to ineffective MTTP, there is reduced chylomicron and VLDL synthesis and secretion.

 Reduced chylomicron and VLDL synthesis leads to severely reduced absorption of dietary fats and fat-soluble vitamins. Accumulation of lipids within enterocytes can be seen on intestinal biopsy.

 Clinical findings present in the first few months of life and include failure to gain weight and grow, steatorrhea, abnormal star-shaped red blood cells

(acanthocytosis), ataxia, and night blindness.

Cholesterol

0

Synthesis occurs in liver, intestines, adrenal glands, reproductive organs. Rate-limiting step is catalyzed by HMG-CoA reductase.

less  HMG-CoA reductase converts HMG-CoA to mevalonate.

 Pharmacology Correlate:

HMG-CoA reductase is the enzyme targeted by statin drugs, which act by competitive inhibition.

 Regulation:

HMG-CoA reductase expression is inhibited by ↑ cholesterol levels. HMG-CoA activity is ↓ by AMP dependent phosphorylation and glucagon. HMG-CoA activity is ↑ by insulin.

ACAT (acylcoenzyme A: cholesterol acyltransferase) esterifies cholesterol inside the liver (for storage) and intestine (for transport in chylomicrons). Free cholesterol in the bloodstream is esterified by LCAT (lecithin-cholesterol acyltransferase):

less  LCAT is bound to plasma HDL and LDL. As it esterifies cholesterol in HDL, it

allows HDL to participate in reverse cholesterol transport.

 Esterification makes the cholesterol more hydrophobic → sequestered in the core of the lipoprotein.

 CETP (cholesteryl ester transfer protein) facilitates the transfer of cholesteryl ester (added by LCAT) from HDL to VLDL, IDL, and LDL in exchange for

triacylglycerol.

SLOS (Smith-Lemli-Opitz-Syndrome) is an autosomal recessive deficiency in DHCR-7 (DHCR-7-dehydrocholesterol reductase), an enzyme used in the synthesis of cholesterol → ↓ cholesterol levels.

(27)

 Symptoms include abnormal facial features, mental retardation and characteristics of autism, and congenital malformations.

 Treatment: Cholesterol supplementation via synthetic cholesterol or dietary efforts may help.

Gluconeogenesis

0

Gluconeogenesis occurs predominantly in the liver, but enzymes are also found in the kidney and intestinal epithelium

less  Muscle cells cannot participate in gluconeogenesis because they lack

glucose-6-phosphatase

Consumes 6 ATP equivalents (4 ATP and 2 GTP) per glucose at a time when energy is already low (starvation, prolonged anaerobic respiration) and often occurs concurrent with ketosis

The 3 regulated steps of glycolysis are essentially irreversible, so different enzymes are required to regenerate glucose. Recall that the 3 irreversible steps of glycolysis are:

less  1) Glucose → G6P

2) F6P → F1,6BP 3) PEP → pyruvate

 These reactions are irreversible using glycolytic enzymes; however,

using differentenzymes, gluconeogenesis makes the reverse of the above reactions energetically possible

Pyruvate to PEP requires 2 enzymes: pyruvate carboxylase and PEP carboxykinase Pyruvate carboxylase activates biotin (vitamin B7), then transfers CO2 from

activated biotin to pyruvate, yielding OAA (oxaloacetate). This occurs in the mitochondria

less  Biotin activation requires 1 ATP (2 per glucose)

 Allosterically stimulated by acetyl-CoA

 Note that OAA can also directly enter the citric acid cycle OAA transported to the cytosol using the malate shuttle

(28)

 Note: since OAA itself cannot transverse the mitochondrial membrane, it is first reduced to malate → when malate enters the cytosol, it is converted back to OAA by malate dehydrogenase

PEP carboxykinase converts the OAA to PEP in the cytosol

less  1 GTP is consumed in this reaction (2 per glucose)

Glycolytic enzymes are used in reverse, until F1,6BP is reached

Fructose-1,6-bisphosphatase dephosphorylates F1,6BP to F6P in the cytosol The final reaction occurs in the smooth endoplasmic reticulum, when G6P is dephosphorylated into glucose by glucose-6-phosphatase → sequestration of this enzymes assures it does not compete with hexokinase during glycolysis

(29)

(30)

Glycogen

0

Glycogen is a highly branched glucose polymer used as the main storage form of glucose in the body.

less  The linear bonds between glucose molecules in glycogen are α-(1,4)

glycosidic linkages; the branching bonds are α-(1,6) glycosidic linkages.

 Glycogen is synthesized and stored primarily in the cytoplasm of hepatocytes and skeletal muscle cells.

 Glycogen can provide energy for about one hour of exercise, depending on intensity.

Glycogen synthesis is the creation of a large glucose polymer. The synthesis of glycogenrequires energy in the form of ATP and UTP.

less  The first step in glycogen synthesis uses ATP to make glucose-6-phosphate

(G6P) from glucose.

 The enzyme that converts glucose to glucose-6-phosphate is either hexokinase orglucokinase.

 Hexokinase and glucokinase both catalyze the same reaction (glucose → glucose-6-phosphate). The difference is that glucokinase is found in liver and pancreas cells and becomes active at higher levels of glucose, while hexokinase is found in most cells and efficiently uses low levels of glucose.

In the second step of glycogen synthesis, glucose-6-phosphate is “mutated” to glucose-1-phosphate (G1P).

less  The enzyme that “mutates” glucose-6-phosphate to glucose-1-phosphate

isphosphoglucomutase.

In the third glycogen synthesis step, glucose-1-phosphate + UTP → UDP-glucose + 2Pi.

less  The enzyme for the conversion of glucose-1-phosphate to UPD-glucose

is UDP-glucose pyrophosphorylase.

The rate-determining step of glycogen synthesis is the addition of UDP-glucose to a pre-existing glycogen chain by glycogen synthase.

less  The enzyme that catalyzes the addition of UDP-glucose to an existing

glycogen molecule is glycogen synthase. Glycogen synthase adds UDP-glucose in an α-(1,4) glycosidic linkage.

(31)

 Glycogen synthase is activated by insulin and glucose and inhibited by glucagon and epinephrine.

In cases where there is not already a pre-existing glycogen molecule, glycogenin is needed. Glycogenin is an enzyme that self-catalyzes the attachment of the first few UDP-glucose molecules to itself. Glycogenin remains at the center of the glycogen molecule.

Branching enzyme makes branch points in a linear glycogen molecule using α-1,6 glycosidic bonds, about every 10 glucosyl residues.

Glycogenolysis is the breakdown of glycogen. The end result is free glucose (liver) or glucose-6-phosphate (muscle).

The first (and rate-determining) step of glycogenolysis is: Glycogen + phosphate → glycogen (less 1 residue) + glucose-1-phosphate.

less  The enzyme that catalyzes the rate-determining step of glycogenolysis

is glycogen phosphorylase.

 Glycogen phosphorylase is activated by epinephrine, glucagon, AMP, and calcium and inhibited by insulin and ATP.

 Glucagon and epinephrine activate glycogen phosphorylase in a G-protein → Adenylyl Cyclase → cAMP → Protein Kinase A → phosphorylase kinase pathway.  Calcium activates glycogen phosphorylase via a calcium-calmodulin complex

in muscle so that glycogenolysis is coupled with muscle contraction.  Insulin deactivates glycogen phosphorylase by activating a protein

phosphatase that dephosphorylates glycogen phosphorylase.

The next step in glycogenolysis is that 1-phosphate is converted to glucose-6-phosphate.

less  Glucose-1-phosphate is converted to glucose-6-phosphate in glycogenolysis

by the enzyme phosphoglucomutase.

 The glucose-6-phosphate may then be used in glycolysis, the HMP shunt, or converted to free glucose (liver).

Glucose 6-phosphatase removes the phosphate from glucose-6-phosphate to create free glucose.

less  In the liver, glucose 6-phosphatase is expressed, which leads to free glucose

being made from glucose-6-phosphate and sent into the bloodstream. In muscle, however, phosphatase is NOT expressed, which means that glucose-6-phosphate is trapped in muscle cells for their own use.

When 4 glucose residues are left on a branch (also known as a limit

dextrin), debranching enzyme moves 3 residues to the end of another branch, leaving one residue with alpha-1,6 linkage. The debranching enzyme then hydrolyzes this remaining residue to yield one free glucose molecule.

(32)

Heme

0

Heme synthesis starts in mitochondria with succinyl-CoA from the citric acid cycle. All the steps in heme synthesis are as follows (Steps critical to know for Step 1 are bolded):

less  δ-aminolevulinate acid(ALA) is synthesize from glycine and

succinyl-CoA via the enzyme ALA synthase in the mitochondrial matrix.

 ALA → porphobilinogen via the enzyme ALA dehydratase in the cytosol  Porphobilinogen → hydroxymethylbilane via the enzyme

porphobilinogen deaminase (aka uroporphyrinogen I synthase)  Hydroxymethylbilane → uroporphyrinogen III via the enzyme

uroporphyrinogen III synthase

 Uroporphyrinogen III → coproporphyrinogen III via the enzyme uroporphyrinogen decarboxylase

 Coproporphyrinogen III → protoporphyrinogen III via the enzyme coproporphyrinogen oxidase

 Protoporphyrinogen III → protoporphyrin IX via the enzyme protoporphyrinogen oxidase

 Protoporphyrin IX combines with Fe 2+ → heme via the enzyme ferrochelatase in the mitochondrial matrix

The first and last 3 steps of heme synthesis occur in mitochondria, the rest occur in the cytosol.

ALA synthase has important regulation: -Activated by P450 inducers

-Inhibited by hemin, glucose -Requires vitamin B6 as a cofactor

Disruption at 4 points in the pathway leads to accumulation of intermediates → porphyrias

Acute intermittent porphyria: autosomal dominant defect of PB-deaminase

less  Characterized by attacks of peripheral/autonomic neuropathies with

intermittent, symptom free periods. It is unclear how porphyrin intermediates cause these symptoms.

(33)

 Symptoms can include abdominal pain (severe and several days' duration), seizures, tachycardia, hypertension, coma, dark or reddish-brown urine,

and psychiatric manifestations (e.g. depression).

Porphyria cutanea tarda: autosomal dominant defect of UROD (uroporphyrinogen decarboxylase)

less  80% of cases are acquired, 20% genetic (autosomal dominant).

 Episodes are precipitated by hepatotoxic agents (e.g. EtOH, hepatitis)

 Initial symptoms: fragile skin that blisters with minimal sun exposure. Patients may also present with pink or dark colored urine, but this is more commonly seen with Acute Intermittent Porphyria.

 Pharmacology Correlate:

Barbiturates (and other inducers) induce the P450 system → consumption of heme → loss of heme's negative feedback on ALA synthase (the rate limiting step of heme biosynthesis) → accumulation of heme intermediates.

Accumulation of these intermediates causes acute exacerbations of porphyrias. Lead poisoning: symptoms primarily via inhibition of ALA dehydrogenase (also called ALAdehydratase or PBG synthase) but also inhibits ferrochelatase

less  Causes microcytic anemia

 Inhibition of ALA dehydratase → accumulation of ALA; if ferrochelatase is inhibited, protoporphyrin IX accumulates.

 Symptoms can closely mimic those of acute intermittent porphyria, or can be more subtle (loss of developmental milestones, temperamental lability). Lead

poisoning in children → encephalopathy, seizures, mental deterioration.

Heme Degradation: Heme → biliverdin → unconjugated bilirubin, transported to liver

Urea Cycle

0

Urea cycle: series of reactions that occur in the liver in order to convert toxic ammonia (product of amino acid catabolism) to urea (molecule with two amine groups)

less  Failure of the urea cycle (mainly genetic vs. cirrhosis) leads to

(34)

Symptoms of hyperammonemia include: Vomiting

Lethargy

Neurological symptoms: flapping hand tremor, ataxia, intellectual impairment, seizures, behavioral changes, eventual coma and death if not corrected

 Excess ammonium (NH4) depletes α-ketoglutarate (α-ketoglutarate + NH4 → glutamate), which leads to inhibition of the TCA cycle

 Treatment for hyperammonemia: limit protein intake or administer benzoate or phenylbutyrate to ↓ ammonia levels

The first step, the conversion of CO2 and ammonia to carbamoyl phosphate, is rate-limiting

less  Enzyme: carbamoyl phosphate synthetase I

Allosteric Activator: N-acetyl-glutamate  This step consumes 2 ATP

 This step takes place in the mitochondria

 The ammonia in this reaction provides the 1st amine groups in urea  Note: HCO3- + NH4+ is equivalent to CO2 + NH3 + H2O

The carbamoyl moiety is transferred to the non-proteinogenic amino acid ornithine to formcitrulline

less  Takes place in mitochondria, then Citrulline is transported into the Cytoplasm  Enzyme: ornithine transcarbamylase

 Most common inherited urea cycle disorder is a deficiency of ornithine transcarbamylase (X-linked recessive unlike the other urea cycle enzyme deficiencies which are autosomal recessive)

X-linked disorder more common in males∴

 Signs and symptoms of ornithine transcarbamylase deficiency: - often evident in the first few days of life (but may appear later)

- ↑ orotic acid in blood and urine (excess carbomyl phosphate converted to orotic acid, a pyrimidine synthesis intermediate)

- ↓ BUN (no urea produced due to enzyme deficiency)

- symptoms of hyperammonemia (should be distinguished from orotic aciduria which has ↑ orotic acid with no hyperammonemia)

(35)

Aspartate condenses with citrulline to form argininosuccinate

less

 The reaction is catalyzed by the cytosolic enzyme argininosuccinate

synthetase

 The amine group on aspartate provides the 2nd amine found in urea  Requires the cleavage of ATP → AMP + PPi

Arginosuccinate is cleaved into arginine and fumarate by argininosuccinate lyase less  Fumarate → malate → oxaloacetate

(this series of reactions occurs in the cytosol and generates a reduced NADH)  Oxaloacetate is converted to aspartate by transaminases, ensuring that the

flow of nitrogen into the cycle is maintained

Arginase I catalyzes hydrolysis of arginine to yield urea and regenerate ornithine

Pyruvate Kinase Deficiency

0

Autosomal recessive mutations of PKLR gene → pyruvate kinase deficiency, which is characterized by three things: 1. Chronic hemolysis 2. increased levels of 2,3-BPG and 3. Lack of Heinz bodies

less  1) Chronic hemolysis (both extravascular hemolysis and intravascular

hemolysis occur)

 2) ↑ levels of 2,3-BPG (2,3-bisphosphoglycerate, also known as 2,3-DPG, or 2,3-diphosphoglycerate) ↓ oxygen affinity of hemoglobin∴

 3) Lack of Heinz bodies within RBCs on peripheral blood smear—vs. G6PD deficiency, a disease in which Heinz bodies are characteristic findings

Mature RBCs have no mitochondria RBCs rely entirely on anaerobic glycolysis ∴ for ATPproduction. Pyruvate kinase deficiency → ↓ levels of ATP, which leads to:

less  ↓ Na/K ATPase activity → Na+ accumulates intracellularly, drawing water

along with it →osmotic fragility, swelling, and intravascular hemolysis of RBCs  Swelling causes RBCs to lose their biconcave shape → chronic

extravascular hemolysis in the spleen

Patients can survive on reticulocytes (still have organelles and can still perform oxidative phosphorylation) and ↑ 2,3-DPG levels (allows an easier release of oxygen from an already low number of RBCs)

(36)

In laboratory testing, addition of ATP to an affected RBC sample will prevent hemolysis

ATP

0

Energy of ATP (the primary currency of cellular energy) is released when two high-energy phosphate bonds are hydrolyzed to form ADP or AMP and free phosphate group

One molecule of glucose that undergoes glycolysis produces two molecules of pyruvate, withnet production of 2 molecules of ATP and 2 molecules of NADH

less  Energy investment phase: the conversion of glucose to F1,6BP

(fructose-1,6-bisphosphate) requires 2 ATP

 Energy production phase: degradation of F1,6BP to 2 molecules of pyruvate yields 4 ATPand 2 NADH

 Glycolysis does not require oxygen but under anaerobic conditions,

converting pyruvate to lactate oxidizes NADH to NAD+, allowing glycolysis to continue Aerobic respiration yields 30-32 ATP per glucose

less  This can be confusing because theoretical calculations yield 36-38 ATP per

glucose; however, pyruvate, ADP, and phosphate need to be transported into the mitochondria and this requires energy

Cysteine Metabolism

0

Cysteine: synthesized from homocysteine and serine, requiring pyridoxine (B6) as a coenzyme

Homocystinuria: several different enzyme defects (all forms are autosomal recessive) that lead to elevation of homocysteine

(37)

 Classical homocystinuria (most common cause of

homocystinuria): deficiency of cystathionine synthase → can be treated with ↓ methionine and ↑ cysteine intake with supplementation of B12 and folate

 Decreased affinity of cystathionine synthase for pyridoxal phosphate → can be treated with high does of pyridoxal phosphate (vitamin B6) to overcome the decreased affinity

 Homocysteine methyltransferase deficiency: defect in the enzyme responsible for the regeneration of methionine from homocysteine

 Methylenetetrahydrofolate reductase (MTHFR) deficiency: defect in the enzyme that catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, a cosubstrate for homocysteine remethylation to methionine  Clinical findings include: mental retardation, atherosclerosis leading to

vascular thrombosis, long thin extremities with eunuchoid features (reminiscent of Marfan's habitus), lens dislocation, and osteoporosis

Note: patients with Marfan's syndrome usually have a lens that is dislocated in an up-and-out direction, while patients with homocystinuria have lens dislocation in a down-and-in direction

Acquired homocysteinemia: seen in both B12 and folate deficiency (both are needed for the remethylation of homocysteine to methionine)

less  Homocysteine is thought to promote atherosclerosis → explaining vascular

disease in patients with homocystinuria and elevated cardiovascular disease risk in people with B12or folate deficiency

Cystinuria: defect in cystine transport proteins in renal proximal tubule and small intestine → ↑ urine levels of cystine

less  Cystine precipitates in acidic (less than pH 7.5) urine → patients suffer from

recurrent cystine renal stones (cystine staghorn calculi)

 Hexagonal or benzene crystals in the urine are pathognomonic of cystinuria  Same amino acid transporter is responsible for the dibasic amino acids

(ornithine, lysine, and arginine)

Mnemonic is COAL → Cysteine, Ornithine, Arginine, Lysine

 Treatment: hydration and alkalization of the urine using potassium citrate or acetazolamide

Lipid Digestion & Metabolism

0

Dietary fat in the intestinal lumen stimulates the secretion of bile and pancreatic enzymes →bile emulsifies dietary lipids → increases the surface area available for pancreatic enzymatic digestion.

less  Pancreatic lipase, colipase, and cholesterol esterase degrade lipids into 2-MG

(monoglyceride) + FAs (fatty acids) + cholesterol which are absorbed by intestinal epithelial cells, then re-esterified into TGs (triglycerides) and CEs (cholesterol esters) and packaged into chylomicrons.

 Chylomicrons contain: 1. TGs (90% of chylomicron) 2. CEs

3. Fat-soluble vitamins and other lipids

4. ApoB-48 (an apolipoprotein specific to chylomicrons and chylomicron remnants) — apoB-48 is a truncated form of apoB-100 that lacks the C-terminal LDL receptor Chylomicrons are secreted by intestinal epithelial cells (due to apoB-48) and circulate in the lymphatics before entering the venous bloodstream via the thoracic duct. (Recall: the thoracic duct empties into the venous system at the junction of the left subclavian vein and left internal jugular vein.)

After entry into the venous circulation, chylomicrons pick up apoC-II and apoE from circulating HDL

less  apoC-II activates LPL (lipoprotein lipase), which releases FFAs (free fatty

acids) and glycerol → FFAs are subsequently taken up and stored within peripheral tissues (adipose, muscle) while glycerol is taken up by the liver.

 The remaining glycerol and cholesterol in the chylomicron are delivered to the liver and ApoC is returned to HDL— apoE permits uptake of remnants (eg,

chylomicron remnants,VLDL remnants (ie, IDL)) by the liver

Excess fatty acids and carbohydrates are converted by the liver into TGs → hepatocytes subsequently package TGs + apoB-100 to form VLDL (very low-density lipoprotein) → apoB-100 allows the secretion of VLDL from the liver into the bloodstream.

less  Like chylomicrons, VLDL entering the bloodstream picks up apoC-II and apoE

from circulating HDL

 Again, LPL (lipoprotein lipase) releases free fatty acids into peripheral tissues, convertingVLDL to IDL (intermediate-density lipoprotein). IDL is synonymous with VLDLremnants.

(39)

 Note: VLDL cannot be directly converted into LDL. On the other

hand, IDL (VLDLremnants) can either be converted into LDL by hepatic lipase and/or lipoprotein lipase, orIDL can be taken up by the liver.

Presence of apoB-100 and apoE on IDL allows liver to endocytose the IDL (via apoB-100/apoE receptor)

less  Alternatively, LPL and/or hepatic lipase may continue to remove TGs

from IDL, convertingIDL into LDL

 LDL is the primary carrier for cholesterol (needed for plasma membranes)  apoB-100 (the only apolipoprotein component of LDL) mediates the

endocytosis of LDLvia interaction with cell-surface apoB-100 receptors (LDL receptors)

HDL is synthesized de novo in the liver and intestines and secreted into the bloodstream carrying apoA-I, apoC-II and apoE. HDL has 3 functions:

less  1. Pick up and sequester cholesterol in the periphery. HDL contains apoA-I

which activates LCAT (lecithin-cholesterol acyltransferase, aka PCAT (phosphatidylcholine-cholesterol acyltransferase)).

LCAT esterifies cholesterol to HDL, allowing HDL to take cholesterol in the bloodstream and return it to the liver via reverse cholesterol transport.

 2. Transport this esterified and sequestered cholesterol acquired from peripheral tissues and deliver it to the liver and other steroidogenic tissues (eg, adrenals, ovaries, testes).

 3. Carry and donate apoC-II and apoE to chylomicrons and VLDL.

 Cholesterol-rich HDL is endocytosed by the liver via the interaction between apoE on HDLand apoE receptors on hepatocytes.

Note: The cholesterol from cholesterol-rich HDL may also be delivered directly into the cells of other tissues via scavenger receptors (eg, SR-B1) which do not mediate endocytosis of HDL, however the specific mechanisms of such scavenger receptor-mediated cholesterol uptake pathways remain incompletely elucidated.

RCT (reverse cholesterol transport): process by which HDL carries cholesterol to the liver for excretion.

less  1. Transfer of cholesterol from peripheral cells to HDL is mediated by the

protein ABCA1. Hereditary defect in the ABCA1 gene leads to a condition called Tangier disease, which is characterized by complete absence (homozygotes) or markedly decreased levels (heterozygotes) of serum HDL.

 2. Esterification/sequestration of cholesterol in HDL occurs by LCAT, as described above.

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 3. Cholesterol-rich HDL is endocytosed by the liver via the interaction between apoE onHDL and apoE receptors on hepatocytes.

The protein SR-B1 also causes cholesterol transfer from HDL to hepatocytes, though not through endocytosis of HDL.

 4. In the liver, cholesterol may be converted to bile and excreted.

 CETP (cholesterol ester transfer protein) catalyzes the transfer of cholesterol from HDL toVLDL, and TGs from VLD to HDL → CETP activity is linked to increased cholesterol levels and coronary artery disease.

Glycolysis

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Occurs in the cytosol and has 2 phases: preparatory (requires ATP) and payoff (generatesATP)