MCB 450 Exam 3

Which statement best describes the direction a chemical reaction will follow? - The concentration of all of the substrates must be higher than that of all of the products for the reaction to proceed in a forward direction. - The enzyme for the reaction must be working at >50% of its maximum efficiency for the reaction to proceed in a forward direction. - Under standard conditions, a reaction will proceed in a forward direction if the free energy ΔG°' is positive. - The direction of a reaction is independent of the initial substrate and product concentrations, because the direction is determined by the change in free energy. - A reaction with positive free energy will proceed in a forward direction if the substrate concentration is raised high enough.

- A reaction with positive free energy will proceed in a forward direction if the substrate concentration is raised high enough.

ATP is organized in ATPase enzyme pockets by multiple interactions including: - The Walker B motif organizes the alpha-phosphate. - Water binds the gamma-phosphate. - Arginine polarizes the gamma phosphate. - Arginine polarizes the nucleophilic water. - All of the above are correct

- Arginine polarizes the gamma phosphate.

Biochemical basis for Arsenite poisoning:

-Arsenite aka mercury inhibit PDC by inactivating the dihydrolipoamide component of the transacetylase enzyme -2,3-dimercaptopropanol (a sulfhydryl reagent), relieves the inhibition by forming a complex with the Arsenite that can be excreted -It significantly inhibits other enzymes using similar enzyme, and reduces overall energy production. -It can also poison other rxn's, serving as a molecular analogue of phosphate and competing with it

Step 1 in FA synthesis using FA synthase:

1) Acetyl CoA + ACP-SH is converted into Acetyl-S-ACP + CoA Acetate is transferred from CoA to the SH group of ACP Specific enzyme used is acetyl CoA-ACP acetyl transferase

Energy Investment Phase with actual molecules being broken down

1) Phosphorylation of glucose: glucose + ATP -> glucose-6-phosphate + ADP 2) Isomerization: G6P -> fructose-6-phosphate 3) 2nd phosphorylation: F6P + ATP -> fructose 1,6-bisphosphate + ADP 4) Cleave of fructose 1,6-BP: fructose 1,6-BP -> glyceraldehyde-3-phosphate + dihydroxyacetone phosphate 5) Isomerization of DHAP: DHAP -> G3P (2)

Structural basis of high phosphoryl-group transfer potential of ATP

1) electrostatic repulsion of negatively charged phosphate groups Like charges repel each other, breaking ATP into ADP relieves the repulsion 2) resonance stabilization Orthophosphate (Pi) has 4 resonance structures 3) stabilization due to hydration H2O binds better to ADP + Pi than to ATP

Step 7: Synthesis of 3-phosphoglycerate -S and P: -Important points: -Enzyme:

1,3-BPG (2) + 2ADP -> 3-phosphoglycerate (2) + 2ATP Reversible step. First substrate level phosphorylation that produces 2ATP (using kinase substrate donor). Enzyme is phosphoglycerate kinase.

Gluconeogenesis enzyme differences of 3 irreversible steps:

1. It begins in the mitochondria, you start with 2 pyruvates and need to get PEP. You have to first convert pyruvate to oxaloacetate using the enzyme pyruvate carboxylase and 2ATP. You then convert the oxaloacetate to phosphoenolpyruvate using PEP carboxykinase and 2GTP. 2. In the step of converting fructose 1,6-bisphosphate to fructose-6-phosphate, you must use the enzyme fructose 1,6-bisphosphatase-1 and H2O. 3. In the final step of converting G6P to glucose, you must use the enzyme glucose-6-phosphatase and H2O.

Precipitating factors of hemolytic anemia in G6PD deficiency: -oxidant drugs -favism -infection

1. oxidant drugs 2. favism (from oxidizing agents in food, such as fava beans which have vicine that can lead to ROS and damage cells); ppl with low G6PD are especially susceptible to vicine toxicity 3. infection (from oxidants produced during inflammation) also associated with neonatal jaundice

The full degradation of a fatty acid chain (26:0) producing 13 molecules of acetyl-CoA requires how many β-oxidation cycles?

12 β-oxidation cycles

The highest energy phosphate bond(s) in ATP is located between which of the following groups:

2 Phosphate groups

How many protons and electrons can the following NAD+ accept?

2 e- and 1 H+

TCA cycle reaction 2: Isomerization of citrate to isocitrate -specific rxn -enzyme

2 step process; first convert citrate to cis-Aconitate using enzyme aconitase Then convert cis-Aconitate to Isocitrate using aconitase again Uses enzyme aconitase, which is a non-heme Iron-sulfur cluster containing protein REVERSIBLE; positive delta G

Step 2 in FA synthesis using FA synthase:

2) Acetyl-S-ACP + Enzyme-SH Acetyl-S-Enzyme + ACP-SH Next, the 2-carbon fragment is transferred to a temporary holding site, the -SH group of the cysteine residue on the enzyme.

Step 9: Dehydration of 2-PG -S and P: -Important points: -problem with enzyme causes what?

2-PG (2) -> phosphoenolpyruvate (2) + 2H2O Reversible step. Delta G is positive. PEP is a high energy enol phosphate. Enzyme is enolase Enolase deficiency is rare but leads to hemolytic anemia

Step 3 in FA synthesis using FA synthase:

3) ACP-SH + Malonyl CoA Malonyl-S-ACP + CoA The now vacant ACP accepts a 3 carbon malonate unit from malonylCoA The specific domain is malonyl CoA-ACP transacylase

Step 8: Phosphate group shift from C3 to C2 -S and P: -Important points: -Enzyme:

3-PG (2) -> 2-phosphoglycerate (2) Reversible step. needs Mg2+. Enzyme is phosphoglycerate mutase.

Step 4 in FA synthesis using FA synthase:

4) Malonyl-S-ACP + Acetyl-S-Enz -> Acetoacetyl-S-ACP + CO2 The Malonyl group loses CO2 in a 4-carbon unit attached to the ACP domain. The domain is 3-ketoacyl-ACP synthase

Step 5 in FA synthesis using FA synthase:

5) Acetoacetyl-S-ACP + NADPH + H+ ---> β-hydroxybutyryl- S-ACP + NADP+ The keto group is reduced to an alcohol. The domain is 3-ketoacyl-ACP reductase

Energy payoff phase with actual molecules being broken down

6) Oxidation of G3P: G3P (2) + 2Pi + 2NAD+ -> 1,3-bisphosphoglycerate (2) + 2NADH + H+ 7) Synthesis of 3-phosphoglycerate: 1,3-BPG (2) + 2 ADP + H+ -> 3-phosphoglycerate (2) + 2ATP 8) Phosphate shift from C3 to C2: 3-PG (2) -> 2-phosphoglycerate (2) 9) Dehydration of 2-PG: 2-PG (2) -> phosphoenolpyruvate (2) 10) Formation of pyruvate: PEP (2) + 2ADP -> pyruvate (2) + 2ATP

Step 6 in FA synthesis using FA synthase:

6) β-hydroxybutyryl-S-ACP crotonyl-S-ACP + H2O A molecule of H2O is removed to introduce a double bond between carbon 2 & 3. The domain is 3-hydroxyl-ACP dehydratase

Oxidative phase of PPP: Step 3 -Substrate and products -Enzyme -generates NADPH

6-gluconoate is then converted in ribulose-5-phosphate Uses enzyme 6-phosphogluconate dehydrogenase Produces a second molecule of NADPH and CO2

Oxidative phase of PPP: Step 2 -Substrate and products -Enzyme

6-phosphogluconolactone is then converted into 6-phosphogluconate Uses enzyme 6-phosphogluconolactone hydrolase (aka lactonase) Rxn is irreversible but not rate-limiting

Step 7 in FA synthesis using FA synthase:

7) crotonyl-S-ACP + NADPH + H+ ---> butyryl-S-ACP + NADP+ The double bond is reduced. The domain is Enoyl-ACP reductase

The electrons released during oxidation reactions cannot be captured by: NAD+ ATP NADP+

ATP

Which of these compounds is oxidized by a multienzyme complex that requires five different coenzymes?

Acetyl CoA

TCA cycle reaction:

Acetyl CoA has 8 electrons which are transferred in the cycle to either NAD+ or FAD Creates 1 GTP, 3 NADH, 1 FAD (2H) Generates 10 ATP total in ETC Ac-CoA + 3NAD+ + FAD + GDP + Pi +2H2O -> CoA-SH + 3NADH + 3H+ + FADH2 + GTP + 2CO2

TCA cycle reaction 1: Synthesis of citrate from acetyl-coA and oxaloacetate -specific rxn -enzyme

Acetyl-CoA + Oxaloacetate -> Citrate H2O gets converted to CoA-SH Uses enzyme citrate synthase IRREVERSIBLE STEP; neg. delta G

Fluoracetate poisoning

Aconitase is inhibited by fluoroacetate, a compound used in rat poison. It gets converted to fluoroacetyl-CoA, whcih condenses with oxaloacetate to form fluorocitrate, a potent inhibitor of Aconitase, resulting in citrate accumulation.

Regulation of PFK-1: -activators

Activated by AMP, ADP, fructose 2,6-bisphosphate -AMP reverses inhibition by ATP, making it a potent activator. It is very sensitive to AMP regulation. -AMP is created by muscle contractions hydrolyzing ATP to ADP, and to meet demand for ATP, cells use the adenylate kinase rxn (combines 2ADP to make ATP and AMP, but more AMP than ATP). -fructose 2,6-bisphosphate is also an activator (see next flashcard)

Overall reaction of fatty acid synthesis:

After 7 cycles, palmitoyl-S-ACP is produced and palmitate is released by palmitoyl thioesterase producing a fully saturated molecule. 8 acetyl CoA + 14 NADPH + 14H+ + 7ATP -> palmitate + 8CoA + 14 NADP+ + 7ADP + 7Pi + 7H2O *The CO2 added to acetyl CoA to form malonyl CoA is released again in the condensation step of fatty acid synthesis.

Oxidative phase of PPP: Step 1 -Substrate and products -Enzyme -generates NADPH

After making G6P from glucose, the G6P is shuttled to the PPP. G6P is converted into 6-phosphogluconolactone Enzyme used is glucose-6-phosphate dehydrogenase (G6PD) This generates NADPH (by reducing NADP+), and NADPH inhibits this enzyme This is a primary regulation step

More on aldolase: -3 types (A,B,C)

Aldolase A: present in muscle, RBC, and most other tissues Aldolase B: present only in the liver. This is the only form that can cleave F-1-BP into DHAP and glyceraldehyde Aldolase C: present in the brain.

Importance of aldolase B

Aldolase B plays big role in fructose metabolism, which occurs in the liver, renal cortex, and small intestinal mucosa. It is the only enzyme that can catalyze F1P breakdown into glyceraldehyde and DHAP, and then allow it to be used in glycolytic pathway.

Glucose to fructose via sorbitol:

Aldose reductase reduces glucose to sorbitol (a sugar alcohol). Sorbitol can then be oxidized to fructose via sorbitol dehydrogenase (present in liver, ovaries, sperm and seminal vesicles).

Isocitrate dehydrogenase: -activators? -inhibitors? -NAD+/NADH ratio?

Allosterically activated by: ADP (a low-energy signal) and Ca2+ Inhibited by: ATP and NADH, whose levels are elevated when the cell has abundant energy stores. ↑ NAD+/NADH speeds up the cycle where as ↓ NAD+/NADH slows the cycle.

Anaplerotic pathways of the TCA cycle: (refilling intermediates in the cycle) Oxaloacetate

As the amount of oxaloacetate is depleted through the efflux of TCA cycle intermediates, the rate of Citrate Synthase reaction decreases and causes the acetyl-CoA conc. to increase, activating Pyruvate carboxylase to produce more oxaloacetate.

A 30-year-old man has been fasting for religious reasons for several days. His blood glucose level is now about 60% of its normal value, but he does not feel lightheaded because his brain has reduced its need for serum glucose by using which of the following substances as an alternate energy source?

Beta-hydroxybutyrate

Which of the following would be associated with HIGH glycolysis rate?

Binding of insulin to its receptor

Changes in blood fuels during fasting

Blood glucose drops after 8 hours of not eating, but then it stabilizes over 40 days of fasting. There is big increase in ketone bodies with fasting. Protein is the last resort to metabolize after prolonged fasting.

CPT1 and CPTII functions:

CPT1: transfers fatty acyl coA across the outermitochondrial membrane from the cytosol CPTII transfers fatty acylcarnitine across inner mitochondrial membrane into the matrix of the mitochondria

Regulation of TCA Cycle: Ca2+

Ca2+ is a positive regulator of both isocitrate dehydrogenase and a-ketoglutarate dehydrogenase

Long-chain fatty acids are shuttled into the mitochondrion bound to what molecule?

Carnitine

Transport of FA-CoA derivatives* into mitochondria for oxidation requires the amine carnitine. -what is carnitine? -where is it found in the body?

Carnitine is a quartenary amine synthesized from the amino acids lysine and methionine and is made in the liver and kidneys. Can also be obtained from the diet (but is not necessary). Skeletal muscle has the high-affinity carnitine transporters and most of the carnitine in the body is stored in skeletal muscle.

Hereditary fructose intolerance (HFI): -etiology? -which specific processes it affects? -symptoms? -treatment?

Caused by deficiency in aldolase B, which cleaves F1P into glyceraldehyde and DHAP. F1P accumulates, making intracellular Pi and ATP levels fall, which compromises gluconeogenesis and liver protein production. AMP accumulates and is degraded, causing hyperuricemia. Symptoms are hypoglycemia, vomiting, jaundice, bleeding, hepatomegaly, renal dysfunction, hyperuricemia and lacticacidemia. Eating fructose, sucrose and sorbital can cause liver failure and death, so treatment is to avoid fructose and sucrose in the diet.

Classic galactosemia:

Causes galactosemia and galactosuria, vomiting, diarrhea and jaundice. Accumulation of G1P and galacticol in nerve, lens, liver and kidney tissues causes liver damager, severe mental retardation, and cataracts.

The hallmarks of acute alcoholism without food intake are acidosis and hypoglycemia. These metabolic changes are due to the:

Cellular metabolism of alcohol generates a high ratio of NADH/NAD, which shuts down liver gluconeogenesis. The body increases lipolysis and utilizes fat as an energy source that produces large amounts of ketone bodies and lactate.

Cataplerotic reactions of the TCA cycle: (efflux for use in other metabolic pathways)

Citrate can go into fatty acid synthesis a-Ketoglutarate can go into amino acid synthesis (neurotransmitter) Succinyl coA can go into heme synthesis Malate can go into gluconeogenesis And oxaloacetate can go into amino acid synthesis or gluconeogenesis

𝛽-oxidation process: Step 4 Cleavage/Thiolysis

Cleavage Convert B-ketoacyl-CoA into a fatty acyl CoA (that is 2 carbons short now) and an Acetyl CoA using the enzyme thiolase Requires addition of CoA-SH

Role of biotin:

Cofactor that is used in CO2 activation and transfer in carboxylation reactions; as well as synthesis of fatty acids and degradation of valine and isoleucine. It is covalently attached to the pyruvate carboxylase enzyme through an amide linkage in Lys residue It is synthesized by the microflora in our gut and can be eaten in the diet from liver, soybean, nuts, and egg yolks.

Use of Ketone Bodies by Peripheral Tissues

Collectively referred to as "Ketone Bodies" - water soluble, released into blood, and used by many tissues In prolonged starvation, the brain starts to utilize ketone bodies (sparing glucose). The liver lacks enzyme thiophorase and therefore cannot use ketone bodies it generates.

Alanine substrate for gluconeogenesis:

Comes from degradation of muscle proteins (only good for several days), so it is generally preserved for the end as a last resort. Alanine is directly converted to pyruvate through alanine aminotransferase

Structure of fatty acids

Contain a hydrophilic carboxyl group head (ionized at pH 7), and a hydrophobic hydrocarbon chain Unsaturated FA have one or more double bonds (usually cis), typically 3 carbons apart cis double bonds kink the hydrocarbon chain Saturated FA and their derivatives are solid (high Tm) Unsaturated FA and their derivatives are liquid (low Tm), and can be oxidized (become rancid).

Acetyl CoA as high-energy bond storage

Contains thioester which is high energy bond (only 1 resonance structure), making it less stable than oxygen ester (which has 2 resonance structures) Also sulfur has larger atomic size than oxygen

The hormone __________ induces lipolysis, whereas the hormone __________ inhibits the process.

Correct: Glucagon; insulin

The NADPH produced during the PPP is the reducing power for the synthesis of:

Correct: Glutathione

One of the main role of the liver is to act as a blood glucose buffer. As such, glucose only enters the glycolic pathway in the liver when blood sugar levels exceed normal levels. Entry of glucose from the blood supply into the liver is regulated by the GLUT2 receptors. Which of the following mechanism is used by GLUT2 receptors to ensure that the liver only uses glucose in times of plenty?

Correct: Higher Km than other Glut receptors

During Glucose 6-phosphate dehydrogenase activity, which reaction takes place:

Correct: The end product is 6-phosphogluconolactone.

ATP is a co-substrate of the enzyme phosphofructokinase (PFK/PFK-1). In most species ATP is also an inhibitor of PFK-1 at higher concentrations. Which statement below would provide a suitable explanation for this discrepancy?

Correct: There are two sites on PFK-1 that bind ATP. One is the active site; the other is the regulatory site where inhibition occurs.

Under anaerobic conditions, the net yield of one mole of glucose is (pick the BEST answer):

Correct: Two moles of lactate and two moles of ATP.

Role of NADPH in fatty acid synthesis:

Cytosolic conversion of OAA to pyruvate generates NADPH Sources of NADPH for FA synthesis are the Pentose phosphate pathway and the malic enzyme reaction that converts malate to pyruvate + NADPH in the cytosol.

Step 5: Isomerization of DHAP and G3P -S and P: -Important points: -Enzyme: -Deficiency with enzyme causes what?

DHAP -> G3P (2) Reversible step. Delta G is positive Marks end of energy investment phase. Enzyme is triose phosphate isomerase (TPI) Deficiency of TPI is rare but causes congenital hemolytic anemia

Deficiency in CPT1 or CPTII associated diseases:

Deficiency in CPTI or CPTII results in hypoglycemia, hypo-ketosis & recurrent episodes of myoglobinuria precipitated by prolonged exercise or fasting. Hypoglycemia results from impaired fatty acid oxidation because the energy requiring steps of gluconeogenesis use NADH and ATP that come from fatty acid oxidation. Between meals, fatty acids are a major source of energy for the liver, and if there is not enough carnitine, there is not enough to allow ketone body synthesis and this impedes gluconeogenesis, which leads to hypoglycemia.

Sources of blood glucose in fed state

Dietary carbs are main source of glucose (carbs -> glucose in the gut). You have increased insulin and decreased glucagon in fed state

Maltose

Disaccharide of 2 glucoses linked by alpha (1,4); broken down by maltase on surface of intestinal epithelial cells into glucose

Isomaltose

Disaccharide of 2 glucoses linked by alpha (1,6); broken down by enzyme isomaltase on surface of intestinal epithelial cells into glucose

Sucrose

Disaccharide of glucose and fructose linked by alpha (1,2); broken down by sucrase on surface of intestinal epithelial cells into glucose

Ketone bodies created during starvation: -How are they created -what drives formation of ketone bodies

During starvation Acetyl-CoA generated from fatty acid metabolism in the Liver is converted into Ketone Bodies Driven by high Acetyl-CoA concentrations in mitochondria ⇒ the levels are dictated by the hormones that cause FA release (glucagon & epinephrine) Rate limiting step of ketogenesis is transcriptionally induced during fasting.

Steps of PDC on E1:

E1 is the catalytic coenzyme TPP (thiamine pyrophosphate), and uses the enzyme pyruvate dehydrogenase. 1) converts pyruvate into hydroxyethyl-TPP by adding pyruvate to TPP and losing CO2 2) Oxidizes hydroxyethyl-TPP and transfers the 2 carbon group to the S on lipollysine, making acyl-lipollysine

Steps of PDC on E2:

E2 is the cat. coenzyme lipollysine, and uses the enzyme dihydrolipoyl transacetylase. 3) Adds CoA-SH to acyl-lipollysine and creates Acetyl CoA which goes on its merry way to TCA

Steps of PDC on E3:

E3 is the cat. coenzyme FAD, and uses the enzyme dihydrolipoyl dehydrogenase 4) The newly reduced lipollysine is oxidized back to lipollysine by FAD, which becomes FADH2 5) e- transfer from FADH2 to NAD+, which gets reduced to NADH

𝛽-oxidation

Each β-oxidation cycle involves four reactions The energy from fatty acids is captured through 𝛽-oxidation in the mitochondrial matrix Very similar pathway to TCA cycle Yields 106 ATP from 1 molecule of palmitate

Symptom of Galactokinase deficiency and Aldolase reductase:

Elevated galacticol can cause cataracts.

In the citric acid cycle, succinate thiokinase (succinyl CoA synthetase) catalyzes the cleavage of the succinyl-CoA thioester bond with formation of a high-energy compound. This compound can then be used by the body in which of the following biochemical pathways?

Elongation of the polypeptide chain ?GTP

Overall 10 steps of glycolysis:

Energy investment phase: 1) phosphorylation of glucose 2) isomerization of G6P to F6P 3) 2nd phosphorylation 4) cleavage of F1,6-BP 5) isomerization of DHAP Energy payoff phase: 6) oxidation of G3P 7) synthesis of 3-PG 8) phosphate shift from C3 to C2 9) dehydration of 2-PG 10) formation of pyruvate

ATPase: -arginine finger -role of Mg2+

Enzyme that catalyzes hydrolysis of ATP and directs the energy where it is needed ATPase activity starts with H2O being organized and deprotonated by Glutamate; making OH- the Nu The arginine finger polarizes the gamma phosphate Mg2+ is required to organize the beta and gamma phosphates of ATP, which shields charge

Pellegra (disease) -etiology -Sx -Relation to other diseases

Etiology: Caused by chronic lack of Niacin (vitamin B3 or nicotinic acid) Symptoms: photosensitive rash, ataxia, diarrhea, neuropsychiatric problems Can also be related to Hartnup's disease (defect in tryptophan absorption) and may also be caused by Crohn's disease (insufficient absorption)

Ariboflavinosis

Etiology: Lack of riboflavin (vitamin B2) causes symptoms similar to Pellegra, causes FADH+ to be half reduced Very uncommon except in protein-energy malnutrition and alcoholism

Fatty acids are oxidized in the muscle and liver:

FA oxidation occurs when blood glucose and insulin are low, and glucagon is increased Fatty acids are activated by fatty acyl-CoA synthetase, which contains a high energy thioester bond and is present on ER, nuclear and plasma membrane

Fatty acids in milk:

FA's with 4 to 10 carbons are found in significant quantities in milk

GLUT5 -role -GLUT5 deficiency?

Facilitated fructose transporter present on apical border of intestinal epithelial cells as well Deficiency of GLUT5 causes fructose malabsorption (aka dietary fructose intolerance)

T/F Acetyl CoA used in fatty acid synthesis is transferred from the mitochondria to the cytoplasm in the form of pyruvate.

False

T/F TPP takes the acetyl group from lipoamide.

False

T/F The PPP produces NADH during the oxidative phase of the pathway.

False

T/F The function of the enzyme acyl CoA synthetase is the reduction of fatty acyl chain.

False

True or False? The enzyme transketolase transfers a three-carbon fragment from a ketose to an aldose.

False

True or false? Glucose 6-phosphate is made in the extracellular space prior to transport into the cytoplasm

False

True or false? If the free energy change (ΔG) of a biochemical reaction is positive, the reaction proceeds spontaneously.

False

True or false? NADP+ inhibits the entry of fatty acyl CoA into the mitochondria as a function of the cell being in the high-energy state.

False

True or false? The carrier of an acyl chain through fatty acid synthesis pathway is serum albumin.

False

True or false? The enamine form of hydroxyethl-TPP is the nucleophile that attacks lipoamide.

False

True or false? The order of the steps involved in the conversion of pyruvate to acetyl CoA is: oxidation, decarboxylation, transfer to CoA.

False

True or false? Fructose-1,6-bisphosphate is a potent allosteric inhibitor of liver phosphofructokinase, which is produced from fructose-6-phosphate by PFK2.

False

True or false? The function of the thioester intermediate formed from glyceraldehyde 3-phosphate is to induce a conformational change that alters the enzyme specificity.

False

True or false? The thiol group of CoA attacks amide bond of acetyllipoamide to create the thioester bond of acetyl-CoA.

False:

TCA break down into 2 stages:

First stage: Reaction 1: Condensation (2C+4C=6C) Reaction 2: Dehydration-Rehydration (Isomerase) Reaction 3: Decarboxylation (C5) (NADH) Reaction 4: Decarboxylation (C4) (NADH) Second stage: Reaction 5: Substrate-level Phosphorylation (GTP) Reaction 6: Oxidation (FADH2) Reaction 7: Hydration Reaction 8: Oxidation (NADH)

Glycolysis:

First step in cellular respiration takes place in cytoplasm starts with glucose and ends with producing 4 ATP overall (2 net), 2 NADH, and 2 molecules of pyruvate

Electron carriers: FAD

Flavin adenine dinucleotide can carry 2 x 1 e- It can carry 2 e- but only 1 at a time, and it is typically involved in oxidation where e- are removed from separate atoms (formation of double bonds) Dangerous molecule because it makes a radical b/c it accepts 1 e- at a time. It is non-soluble and embedded in the active sites of proteins

A 7-year-old girl is brought to the emergency department by her parents with a complaint of severe polyuria. Laboratory examination reveals ketones in her urine. Which of the following is the most likely source of the ketones?

Free fatty acid breakdown

Glycerol substrate for gluconeogenesis:

From degredation of triacylglycerides in adipose tissue. The hormone sensitive lipase is activated by glucagon, epinephrine, and cortisol. Glycerol is converted to G3P by glycerol kinase

Multiple carboxylase deficiency

From of metabolic disorder involving failures of carboxylation enzymes. Results from biotinase deficiency, which cleaves biotin from Lysine preventing recycling of biotin and it is lost to urine. Sx include myalgia, lethargy, anorexia and depression. Treatment is biotin supplementation.

Step 4: Cleavage of fructose 1,6-bisphosphate -S and P: -Important points: -Enzyme: -problem with enzyme causes what?

Fructose 1,6-bisphosphate -> glyceraldehyde-3-phosphate + dihydroxyacetone phosphate Reversible step. Delta G is positive. Specifically uses enzyme aldolase. Genetic mutations in aldolase B causes hereditary fructose intolerance (HFI).

Cleave of fructose 1-phosphate: -what molecules does it make? -what can that new molecule do?

Fructose-1-phosphate is cleaved by aldolase B into DHAP + glyceraldehyde, NOT G3P. DHAP can then enter glycolysis or gluconeogenesis. Glyceraldehyde goes onto either become G3P, glycerol and subsequently either phospho- or triacylglycerols.

Step 3: 2nd phosphorylation -S and P: -Important points: -Enzyme: -deficiency of enzyme causes what?

Fructose-6-phosphate + ATP -> Fructose 1,6-bisphosphate + ADP Irreversible. 1st committed step in pathway. Rate-limiting step and is highly regulated. 2nd ATP is used here. Delta G is negative Enzyme is phosphofructokinase-1 (PFK-1) Deficiency of PFK-1 causes hemolytic anemia

TCA Cycle Reaction 7: Hydration of fumarate into malate

Fumarate is hydrated into malate by enzyme called fumarase (aka fumarate hydratase). It is REVERSIBLE, delta G is negative. Fumarate is also produced by the urea cycle in purine synthesis and during catabolism of the amino acids PHE and TYR

Now that we have palmitate, how do we make elongate it and make other unsaturated FA's?

Further elongation and desaturation of palmitate and dietary FAs (if required) occurs in mitrochondria and ER by desaturases. Human desaturases cannot introduce double bonds farther out than carbon 9. Both α-linoleic acid [18:2(∆9,12)] and α-linolenic acid [18:3(∆9,12,15)- pictured] are essential dietary FA required for the synthesis of eicosanoids, which are precursors to prostaglandins , thromboxanes, leukotrienes, and related compounds.

Step 6: Oxidation of G3P into 1,3-BPG -S and P: -Important points: -Enzyme:

G3P (2) + 2Pi + 2NAD+ -> 1,3-bisphosphoglycerate (2) + 2NADH + H+ Reversible step. Delta G is positive. It reduces NAD+ into NADH. 1,3-BPG is an acyl phosphate which has high phosphoryl transfer potential. This step is technically a 2 step coupled process of "oxidation-phosphorylation." Enzyme is G3P dehydrogenase

Oxidant drugs: Primaquine

G6PD deficiency protects against malaria. The parasites causing this disease require NADPH for optimal growth. Primaquine is a commonly used and highly effective antimalarial drug. However, indiscriminate use of primaquine will cause hemolysis in individuals deficient in G6PD.

Indirect regulation of glucokinase in the liver: -function -what inhibits it -GKRP

GK functions as a glucose sensor for controlling plasma glucose homeostasis (which is typically 5 to 7 mM). It is not inhibited allosterically but indirectly by F6P. GK regulatory protein (GKRP) exists in nucleus of liver cells. In the presence of F6P, GK is translocated in the nucleus bound tightly to GKRP, thus rendering the enzyme inactive. When glucose levels rise, this causes GKRP to release GK, and the enzyme re-enters the cytoplasm where it phosphorylates glucose to G6P.

Galactose metabolism

Galactokinase produces galactose 1-phosphate. In an exchange reaction with UDP-glucose, we can get UDP-galactose which is used in the synthesis of glycoproteins and other biomolecules

Two main types of cellular fuels:

Glucose (is more oxidized; undergoes glycolysis) Fatty acids (is less oxidized, undergoes Beta oxidation)

Step 1: phosphorylation of glucose -S and P: -Important points: -Enzyme:

Glucose + ATP -> Glucose-6-phosphate + ADP Irreversible step. Its traps glucose inside of the cell. 1st ATP is used here It is the start of energy investment phase The delta G is negative Enzyme is hexokinase

Anaerobic glycolysis:

Glucose goes through glycolysis to form pyruvate, but then because there isn't any oxygen the NADH has to be rapidly oxidized to NAD+ to continue glycolysis, and NADH is oxidized to NAD+ by lactate dehydrogenase. Only 2 ATP are produced. Pyruvate becomes lactate and then lactate enters the Cori cycle.

Which one of the following is a common intermediate in the conversion of glycerol and lactate to glucose? Pyruvate Oxaloacetate Malate Glucose 6-phosphate Phosphoenol pyruvate

Glucose-6-phosphate

Step 2: isomerization of G6P -S and P: -Important points: -Enzyme:

Glucose-6-phosphate -> Fructose-6-phosphate Reversible step. Makes aldose into a ketose b/c its easier to split into two 3C molecules. Delta G is positive Enzyme is phosphoglucose isomerase

Process of secreting fatty acid's from liver:

Glycerol 3-P combines with fatty acids-CoA (palimitate that is combined with CoA and made with fatty acid synthase), and becomes triacylglycerols and is transported via VLDL in the lymph vessels

Synthesis of TAG's in liver and adipose tissues:

Glycerol 3-PO4 is produced from glucose in both liver and adipose tissues. Liver has glycerol kinase, so it can directly phosphorylate glycerol to glycerol 3-PO4

Sources of blood glucose in fasting state

Glycogen is the main source of glucose, which is stored in the liver and muscle. Your body undergoes glycogenolysis to get you that glucose from glycogen for about 48 hours. You have decreased insulin and increased glucagon (PKA) at that time.

Tissue interrelationships during fasting:

Glycogenolysis in liver is induced due to low insulin/glucagon ratio. The brain and RBCs use the glucose released by the liver. Adipose releases free fatty acids and glycerol from stored triglycerides. The liver converts FA-derived Ac-CoA to make ketone bodies that are used as energy by the brain and muscles. Protein in the muscle is broken down and amino acids travel to the liver to be used as gluconeogenic precursors. Amino acid metabolism in liver generates urea that travels to the kidneys for excretion. Lactate produced in the RBCs and glycerol made in the adipose return back to the liver for gluconeogenesis.

Glucose 6-P Dehydrogenase deficiency (G6PD deficiency: -etiology -common?

Hereditary deficiency in this enzyme causes hemolytic anemia because of inability to detoxify oxidizing agents (due to insufficient reduced glutathione) It is the most common disease producing enzyme abnormality in humans, it is X-linked so common in females, and very common in the Middle East, Mediterranean, topical Africa and Asia However carriers have increased resistance to malaria

Hexokinase 1: -Role -Kinetics -Regulation by G6P

Hexokinase 1 is specifically present in all tissues, hexokinase IV (glucokinase) is only present in the liver and b-islet cells of the pancreas only. Enzyme used to phosphorylate glucose into G6P. It is an irreversible but regulatory step. It has a low Km (high affinity for glucose) but low Vmax, which permits efficient phosphorylation and subsequent metabolism of glucose even at low concentrations Hexokinase 1 is feedback inhibited by G6P; this inhibition prevents the enzyme from tying up all the intracellular Pi in the form of G6P.

Which of the following would be associated with LOW pyruvate dehydrogenase rate?

High Acetyl CoA

Mammalian pyruvate dehydrogenase complex (PDC) is inactivated by pyruvate dehydrogenase kinase in the presence of :

High NADH

𝛽-oxidation process: Step 2 Hydration

Hydration Convert trans-enoyl-CoA into B-hydroxyacyl-CoA using the enzyme enoyl-CoA hydratase H2O is added

Effects of hyperglycemia on sorbitol metabolism:

Hyperglycemia and adequate NADPH can result in excessive sorbitol accumulation inside certain cells (lens, retina, Schwann cells, liver, kidney and RBC) because insulin is not required for glucose to enter these cells. Sorbitol does not efficiently cross the plasma membrane, so the osmotic effect of excessive sorbitol in these cells can damage the cells in hyperglycemia of uncontrolled diabetes.

A hereditary deficiency in aldolase B leads to which of these conditions?

Hypoglycemia, hyperuricemia, liver failure

Cataplerotic reactions of TCA cycle specifically in the liver: Malate

If glucose is needed (fats are primarily being metabolized in the Liver), then the malate dehydrogenase reaction is inhibited (because NAD+/NADH ratio decreases) malate accumulates, and is exported to the cytoplasm for use in gluconeogenesis.

Cataplerotic reactions of TCA cycle specifically in the liver: Citrate

If glucose is plenty, the ATP is not being used and the NADH levels become too high, which inhibits isocitrate dehydrogenase and a-ketogluatarate dehydrogenase. The excess citrate gets shuttled to the cytoplasm where it is used for fatty acid synthesis. The excess a-ketogluatarate is also used for amino acid synthesis.

Why are children more susceptible to developing ketosis:

In children, the level of ketone bodies can rise much higher than in adults because the fasting state is reached much faster (high muscle/adipose ratio), particularly if vomiting or anorexia (no appetite) occurs.

High energy metabolites in cells

In glycolysis, 1,3-bisphosphoglycerate and PEP (phosphoenolpyruvate) In muscle, creatine phosphate In TCA cycle, Acetyl CoA

Difference between glycolysis and gluconeogenesis:

In glycolysis, glucose is catabolized, in GNG, it is synthesized (or anabolized). ATP is consumed rather than produced in GNG. Lastly, NADH is oxidized to NAD+ instead of NAD+ being reduced.

Sources of blood glucose in starving state

In starved state, once glycogen stores are depleted there are other ways to use precursors to generate glucose, from glycerol, lactate, and amino acids and undergo gluconeogenesis in the liver to form glucose and send it to where it is needed. Between fasting and starved, brain adapts to using ketones (from fatty acids), sparing glucose.

Regulation of TCA Cycle: Regulation of Isocitrate dehydrogenase: I

In the absence of ADP, binding of isocitrate to one subunit increases the binding of isocitrate to the other subunits (positive cooperativity). In presence of ADP, ALL subunits are in active conformation, & isocitrate binds more readily (lowers Km).

Regulation of mitochondrial 𝛽-oxidation: -activators? -inhibitors?

Increased fatty acids activate it Increased AMP turns off fatty acid synthesis (and thus activates B-oxidation) CPT1 is inhibited by malonyl CoA (rate limiting step of FA synthesis) High NADH/FADH2 build-up inhibits it as well

PEPCK is activated by:

Increased glucagon (mainly increased cAMP) and glucocorticoids.

Regulation of PFK-1: -inhibitors

Inhibited by ATP and citrate. -It is allosterically inhibited by ATP, which acts as an "energy rich" signal. ATP binds at the 2nd site away from the catalytic site. -citrate is also an allosteric inhibitor of PFK-1; it signals availability of alternate fuels, such as fatty acids.

a-ketoglutarate dehydrogenase complex -inhibited by? -activated by?

Inhibited by its products, NADH and succinyl CoA Activated by Ca2+, not regulated by phosphorylation like PDH complex

Regulation of the PDH Complex: -inhibitors? -activators?

Inhibited by: Low blood glucose, high ATP, high NADH, and high Acetyl-CoA Activated by: High pyruvate, high NAD+, and high ADP

Pyruvate dehydrogenate complex:

Irreversible step to convert pyruvate into Acetyl CoA Takes place in the mitochondrial matrix. It is a multienzyme complex composed of E1 (thiamine pyrophosphate), E2 (lipoate), and E3 (FAD) which are the catalytic coenzymes Uses stoichiometric coenzymes CoA and NAD+ Uses same mechanism as alpha-ketoglutarate dehydrogenase in TCA cycle

In a metabolic pathway, a reaction that best serves as a committed step: Has to be the last step of the pathway. Is endergonic. Is metabolically irreversible. Can be easily reversible. Is a near equilibrium reaction.

Is metabolically irreversible.

TCA cycle reaction 3: Oxidative-decarboxylation of isocitrate into a-ketoglutarate -specific rxn? -rate limiting? -NAD+ reduction -CO2?

Isocitrate is converted into a-ketoglutarate by isocitrate dehydrogenase. IRREVERSIBLE OXIDATIVE-DECARBOXYLATION; RATE LIMITING STEP Lose 2 e-, yield first of 3 NADH molecules, and first release of CO2

Lactate substrate for gluconeogenesis:

It comes from RBC's, skin, brain and muscle (from the Cori cycle). Lactate is produced through anaerobic cellular respiration in exercising muscles, and cells lacking mitochondria (RBC's, lens of eye). The lactate diffuses into the blood and is taken up by the liver where it then undergoes GNG. Lactate is converted into pyruvate by the enzyme lactate dehydrogenase, and reduces NAD+ in the process.

Why is indirect regulation of glucokinase important?

It decrease futile cycling between glucose and G6P under low-glucose conditions, and decreases the lag between a rise in intracellular glucose and the onset of its phosphorylation.

Why ATP is cellular energy currency

It has a phosphoryl-transfer potential that is intermediate among important phosphorylated biomolecules Resting humans consume about 88 pounds (40 kg) of ATP in a day

GLUT2 -location -role -kinetics

It is a facilitated glucose transporter that transports glucose down its concentration gradient into the cell (located on the opposite side of SGLT1, the basolateral side) It works independent of insulin, and takes nutrients in from liver portal circulation. GLUT2 has a high capacity (Vmax) but low affinity (high Km) for glucose, Km~ 15 mM

Regulation of pyruvate kinase: -inhibitors in all glycolytic tissues including liver

It is allosterically inhibited by: -ATP, acetyl CoA, and long-chain fatty acids (all signs of an abundant energy supply), -accumulation of alanine (that can be synthesized from pyruvate all in one step) The accumulation of fructose 1,6-bisphosphate triggers its activation.

Coenzyme A: associated vitamin?

It is an activated carrier of an acyl group, and contains a component of pantothenate (vitamin 5) which is cofactor for fatty acid synthesis. -Deficiency of pantothenate kinase results in neurological problems

A little more about PFK-1: -3 isoforms?

M PFK-1 (muscle), L (liver), and C (highest level in platelets, kidney and fibroblasts, but present in all tissues). All are tetramers that are allosterically regulated.

Galactose

Main dietary source is the disaccharide lactose (made of galactosyl and glucose). Galactose is released from lactose in small intestine by the digestive enzyme lactase, and enters cells without insulin. Lactose intolerance can result due to deficiency in lactase.

Oxidative phase of PPP:

Main purpose is to make NADPH, which is necessary for biosynthetic reactions. This occurs in ALL cell types, and is the only pathway for generation of NADPH in cells without mitochondria (like RBC). A sudden increase in PPP activity is the fastest known cellular response to oxidative stress by generating NADPH. NADPH can also be made by malic enzyme in cells containing mitochondria. RXN'S ARE IRREVERSIBLE

Non-oxidative phase:

Main purpose is to make ribose-5-phosphate, which can be used to make nucleotides, as well as interconverting sugars. Rxn's are irreversible, and basically allows the conversion of ribulose-5-phosphate into ribose 5-P or other glycolysis intermediates or sugars that can interconvert amongst each other.

TCA Cycle Reaction 8: Dehydrogenation of malate into oxaloacetate

Malate is oxidized to oxaloacetate using enzyme malate dehydrogenase. REVERSIBLE; pos. delta G, rxn produces third NADH.

Alcohol intoxication and hypoglycemia:

Metabolism of EtOH results in increase of NADH conc. in liver, which causes intermediates of gluconeogenesis to be diverted into alternate rxn pathways, resulting in decreased synthesis of glucose. Alcohol impairs gluconeogenesis because pyruvate is not available as a substrate to produce glucose (hypoglycemia) Alcohol impairs conversion of lactate to pyruvate and increases serum lactic acid levels. Alcoholic hyperlipidemia is caused by excess glucagon secretion, and induced by starvation and counter-regulatory hormones.

Walker Boxes:

Mg2+ also connects to Walker A (Threonine) and Walker B (Aspartate), which are the enzyme binding pockets. The association of ATP with the Walker boxes induces conformational change enzyme residues then come close together to interact (proximity and orientation). In some cases, kinase has 2 binding pockets and can transfer the gamma phosphate to another molecule

Fructose

Mostly comes from the disaccharide sucrose (which contains glucose and fructose). Entry of fructose into cells is not insulin dependent, and presence of fructose does not promote insulin secretion GLUT 5 is fructose transporter for intestinal cells GLUT2 is low-affinity bidirectional transporter for glucose and fructose in liver cells.

Creatine phosphate

Muscle cells store energy in the high-energy phosphate bonds of creatine phosphate It has a delta G greater than ADP, so useful for quick energy source for strenous exercise It has many mechanisms to maintain ATP homeostasis

In tissues deriving energy anaerobically (such as rapidly exercising skeletal muscle), lactate, rather than pyruvate, serves as the end product of glycolysis. Which of the following statements best explains why pyruvate is converted to lactate under these circumstances?

NAD+ necessary for the glyceraldehyde phosphate dehydrogenase reaction is regenerated. I

NADPH: -difference from NADH -ratios of reduced to oxidized forms in cytosol for NADH and NADPH

NADP+ differs from NAD+ by single phosphate on one of the riboses Certain enzymes of biosynthetic pathways are NADPH-specific The ratios of reduced to oxidized forms in cytosol of hepatocytes favor reductive biosynthetic role for NADPH, and oxidative role for NAD_ NADPH/NADP+= 10:1 NADH/NAD+= 1000:1

Which of the following is an electron carrier in anabolic pathways?

NADPH

Why does G6PD deficiency result in a propensity for hemolytic anemia?

NADPH production is compromised, resulting in an inability to maintain the cytoplasm in a reducing state

Electron carriers: NAD+

Nicotinamide adenine dinucleotide, can carry 2e- It is stable and soluble It is involved in oxidation of alcohols and aldehydes in which 2 electrons are removed (as a hydride ion (H-))

Why does G6PD deficiency affect RBCs so severely?

Other tissues have alternate pathways for producing NADPH (e.g., malic enzyme in fatty acid biosynthesis) RBCs can make NADPH only via the pentose phosphate pathway Therefore, the G6PD mutation has a much more severe effect on RBC

A 57-year-old man with type 1 diabetes mellitus has progressive retinopathy. Ophthalmoscopic examination reveals macular edema and retinal thickening with hard exudates of yellow-white lipid deposits. Which of the following contributes to hyperlipidemia in type 1 diabetes, and thus the risk for lipid deposition in the retina?

Overactive hormone-sensitive lipase

𝛽-oxidation process: Step 3 Oxidation

Oxidation Convert B-hydroxyacyl-CoA into B-ketoacyl-CoA using the enzyme L-hydroxyacyl-CoA dehydrogenase NAD+ is reduced to NADH+H+

What are the repeated steps of β-oxidation? Assume sat. FA with even number of carbons

Oxidation hydration oxidation thiolysis

𝛽-oxidation process: Step 1 Oxidation

Oxidation Convert fatty acyl CoA into trans-enoyl-coA using the enzyme acyl-CoA dehydrogenase FAD is reduced to FADH2

The oxidation of fatty acids is best described by which of the following sets of reactions?

Oxidation, hydration, oxidation, carbon-carbon bond breaking

Step 10: formation of pyruvate -S and P: -Important points: -problem with enzyme causes what?

PEP (2) + 2ADP -> pyruvate (2) + 2ATP Irreversible step. 2nd step of ATP's formed with second substrate-level phosphorylation. Delta G is neg. Enzyme is pyruvate kinase, and uses high energy phosphoryl group on PEP to drive the rxn. Pyruvate kinase deficiency leads to chronic hemolytic anemia (2nd most common cause of hemolytic anemia).

Hemolytic anemia causes:

PFK-1 deficiency Phosphate isomerase deficiency (rare) causes congenital hemolytic anemia Enolase deficiency (rare) Pyruvate kinase deficiency leads to chronic hemolytic anemia (2nd most common cause)

Which reaction or pathway in the diagram shown would most likely be stimulated by a decrease in hepatic [NADPH]/[NADP+]?

PPP

Which of the following enzymatic steps are not part of the gluconeogenesis pathway? Phosphofructokinase 1 Enolase Phosphoglycerate kinase Phosphoglucose isomerase Aldolase

Phosphofructokinase 1

SGLT1 (sodium-glucase linked transporter 1): -location -role -SGLT1 deficiency?

Present on intestinal lumen side (apical side), is a symporter that transports glucose and galactose against a concentration gradient using energy provided by an electrochemical gradient of sodium

Oxidation of Odd Chained Fatty Acids:

Proceeds using B-oxidation until on Propionyl (3C) CoA remains then biotin and Coenzyme B12 are used to make further modifications, and create succinyl CoA which goes into the TCA cycle

A teenager, concerned about his weight, attempts to maintain a fat-free diet for a period of several weeks. If his ability to synthesize various lipids were examined, he would be found to be most deficient in his ability to synthesize:

Prostaglandins.

Pyruvate dehydrogenase complex (PDC) inhibition

Pyruvate dehydrogenase complex (PDC), the enzyme responsible for converting pyruvate into acetyl CoA for the Kreb's cycle is inhibited during gluconeogenesis. It is inhibited by high levels of acetyl-CoA and NADH from fatty acid oxidation. It is otherwise active during glycolysis (obviously to keep cellular respiration going).

A 50-yr-old man presented with symptoms of weakness, fatigue, and shortness of breath. His hemoglobin levels were low. Red blood cells isolated from the patient showed very low levels of lactate production. Which of the following enzyme deficiency could cause his anemic symptoms?

Pyruvate kinase

A 50-year-old chronic alcoholic presents with dementia, paralysis of lateral gaze, and difficulty walking. The vitamin deficient in this patient is required as a cofactor for which of the following enzymes?

Pyruvatedehydrogenase

Carbohydrates are less-efficient fuel sources than fats because the carbon in fats is more:

Reduced

Fructose 2,6-bisphosphate regulation of PFK-1:

Reduces the inhibitory effect of ATP (makes enzyme less sensitive to ATP inhibition). When there is a large amount of F6P due to TCA backup, PFK-2 will make F6P into F-2,6-bisphosphate in the liver (under hormonal regulation by insulin and glucagon). However, the increased presence of F-2,6,-BP activates PFK-1 and increases its affinity to F6P, and as such activates PFK-1 activity.

Uses of NADPH:

Reductive biosynthesis Maintenance of reduced glutathione Reduction of hydrogen peroxide Cytochrome P450 mono-oxygenase system Phagocytosis by WBC Synthesis of NO (which is a potent vasodilator, neurotransmitter, decreases PLT aggregation, and has a role in macrophage function)

Regulation of TCA Cycle: respiratory control

Respiratory control: Rate of ATP utilization controls both the NAD+/NADH and ADP/ATP ratios in mitochondria. Controls entry of acetyl-CoA in TCA cycle NADH acts as a negative regulator ADP acts as a positive regulator

Sucrase-isomaltase complex deficiency

Results in an intolerance of ingested sucrose, very prevalent in Inuit people Treatment is dietary restriction of sucrose and enzyme replacement therapy

Non-oxidate phase of PPP: -enzyme to form ribose-5-phosphate -enzyme to form xylulose-5-phosphate

Ribulose-5-phosphate converts to ribose-5-phosphate via enzyme ribulose-5-phosphate isomerase. It also converts to xylulose-5-phosphate via enzyme ribulose-5-phosphate epimerase.

Starch: -digestion into oligosaccharides -role of amylase?

Starch is long polymers of glucose linked by linear alpha (1,4) bonds and alpha (1,6) branches The are first broken down by salivary alpha-amylase and then subsequently by pancreatic alpha-amylase Amylase catalyzes the hydrolysis of alpha (1,4) glycosidic bonds, and produce alpha-maltose, alpha-isomaltose, and trisaccharides.

The Citric Acid Cycle/Kreb's Cycle/Tricarboxylic Acid Cycle:

Starts with Acetyl CoA and ends with oxaloacetate Point is to cleave a 2 carbon compound, release the carbons as CO2 and take the electrons using FAD and NAD+ Pyruvate to Acetyl CoA to citrate to isocitrate to alpha-ketoglutarate to succinyl-CoA to succinate to fumarate to malate to oxaloacetate Mneumonic is "Prostitutes Ask: Can I Keep Selling Sex For Money, Officer?"

Fatty acids with physiological importance: -specific names and carbon/double bond numbering

Structural lipids and triacylglycerols contain primarily fatty acids of at least 16 carbons Stearate (18:0) Oleate (18:1(9)) cis-delta9 Linoleate (18:2(9,12)) cis,cis-delta9,12. this molecule is a precursor of prostaglandins. Linolenate (18:3(9,12,15) all cis Names for fatty acids depend on if they are near the carboxy end or the omega end of the chain

A 34-year-old woman presents to her physician complaining of oral ulcers. A careful history reveals that she is a strict vegetarian and does not eat meat, fish, poultry, eggs, or dairy products. She is found to have a severe riboflavin deficiency. The function of which enzymes in the TCA cycle would be most directly affected by the riboflavin deficiency?

Succinate dehydrogenase

TCA Cycle Reaction 6: Dehydrogenation of succinate into fumarate

Succinate is converted into fumarate with enzyme succinate dehydrogenase. FAD gets reduced to FADH2. REVERSIBLE; Delta G is 0 Succinate dehydrogenase is the only enzyme EMBEDDED into mitochondrial membrane to prevent escape of FAD free radicals

A 36-year-old man with viral pneumonia has an episode of hemolysis. Over the next week, he has an increased rate of reticulocytosis. Which of the following compounds serves as a precursor to heme in the reticulocytes?

Succinyl CoA

TCA Cycle Reaction 5: Cleavage of succinyl-CoA into succinate

Succinyl-CoA is converted into succinate by succinyl-CoA synthetase Enzyme succinyl-CoA synthetase also called succinate thiokinase REVERSIBLE (neg. delta G however); also converts GDP+Pi into GTP and releases CoA-SH This rxn is coupled to substrate level phosphrylation of GDP into GTP. Succinyl CoA is also produced from propionyl CoA derived form the metabolism of fatty acids (with odd number of carbons) and amino acids

PEPCK deficiency

Sx include lactic acidemia, loss of muscle tone, hepatomegaly, failure to thrive, and hypoglycemia.

Where and when are fatty acids synthesized?

Synthesis of Fatty Acids (FA) occurs primarily in the liver and lactating mammary gland, less so in adipose tissue FA are synthesized from acetyl CoA derived from excess protein and carbohydrate FA synthesis uses ATP and NADPH as energy sources

Thiamine (vitamin B1) deficiency (aka Beriberi): -role of TPP in rxn's? -symptoms? -wet Beriberi?

TPP dependent reactions are PDC, alpha-ketoglutarate dehydrogenase, and transketolase Symptoms include weight loss, emotional disturbances, tremors, and lactic acidosis It is rare in the USA but does occur in alcoholics (wet Beriberi); called wet because of fluid retention It is the most common cause of congenital lactic acidosis

Thiamine pyrophosphate (TPP) role in transketolase:

TPP is the coenzyme form of vitamin B1 (thiamine). It is a cofactor for transketolase, pyruvate decarboxylate (in the pyruvate dehydrogenase complex), a-ketoglutarate dehydrogenase (TCA cycle), and branched-chain a-keto acid dehydrogenase (of branched-chain amino acid metabolism).

T/F The aconitase reaction isomerizes Citrate to Isocitrate through an alkene intermediate.

TRUE

Pyruvate kinase is a tetrameric enzyme that can exist as an L (found mostly in liver) or M (found mostly in muscles) form. Which of the following statements comparing the L and M forms of pyruvate kinase is true?

The L form is more likely to become phosphorylated under low blood sugar conditions.

Maintenance of reduced glutathione role of NADPH in RBC:

The RBC derives its energy by converting glucose into two molecules of lactate, gaining two ATP 10% of the glucose entering the RBC flows into the pentose phosphate pathway to generate NADPH NADPH is necessary to keep glutathione in the reduced state, in order to maintain membrane integrity

Complete Oxidation of glucose

The electrons from glucose oxidation feed into the electron transport pathway, driving synthesis of ATP.

Role of NADPH in phagocytosis: -enzyme -superoxide radical -myeloperoxidase -ROS

The enzyme NADPH oxidase converts oxygen into superoxide radical O2-, aka oxidative/respiratory burst. This superoxide radical can react with H2O to form H2O2, and it can also use myeloperoxidase to make HOCl (bleach) which are both toxic to microorganisms These are called reactive oxygen species (ROS)

Sources of Acetyl-CoA, a substrate for the TCA cycle:

The fatty acid palmitate Ketone body acetoacetate Sugar, glucose Pyruvate itself And the amino acid alanine or ethanol

The Thioester bond of Acetyl-CoA is favored for hydrolysis due to which of the following?

The lack of resonance between the ketone oxygen and the sulfur atom

Regulation of pyruvate kinase: -inhibitors in only the liver

The liver isozyme (L form) is also regulated hormonally: -Glucagon activates cAMP-dependent protein kinase (PKA), which phosphorylates the pyruvate kinase L isozyme, inactivating it. -When glucagon level drops, a protein phosphatase (PP) dephosphorylates pyruvate kinase, activating it. -This mechanism prevents the liver from consuming glucose by glycolysis when blood glucose is low; instead, the liver exports glucose. -The M (muscle) isozyme is not affected by this.

Hexokinase IV (glucokinase): -location -kinetics -function

The specific hexokinase expressed in hepatocytes (of the liver) and pancreatic beta cells, which is responsible for catalyzing rxn of Glucose to G6P. Glucokinase is a high Km (low affinity) and high Vmax enzyme (high capacity).

Pentose phosphate pathway:

There is an alternative branch off glycolysis to produce the sugars that make up DNA and RNA. This pathway, called the Pentose Phosphate Pathway, is special because no energy in the form of ATP is produced or consumed in this pathway. There is an oxidate phase and non-oxidative phase.

Coenzyme A is an acyl group carrier. The acyl group is linked to coenzyme A via the :

Thioester bond

Second step of FA synthesis using fatty acid synthase:

This enzyme is a multienzyme complex in bacteria and a dimer in animals with 7 activities (aka 7 steps) The growing FA chain is tethered by a 4'-phosphopantetheine group (a component of CoA) to the acyl carrier protein (ACP) subunit The multifunctional fatty acyl synthase molecule has multiple enzymic domains that carry out the various catalytic reactions You repeat the steps 3 to 7 seven times to yield palmitate (16:0)

First irreversible step bypassed during gluconeogenesis: Converting pyruvate to PEP

This first step is technically 2 steps to of carboxylation and decarboxylation that are coupled to drive the rxn. 1) Convert pyruvate to oxaloacetate with enzyme pyruvate carboxylase, 2ATP, CO2, and biotin. 2) Then convert oxaloacetate to PEP using phosphoenolpyruvate and 2GTP using enzyme phosphoenolpyruvate carboxylkinase (PEPCK)

Second irreversible step bypassed during gluconeogenesis: Converting fructose 1,6-bisphosphate to fructose-6-phosphate

This is completed using the enzyme fructose-1,6-bisphosphatase-1 and H2O. Glucagon reduces concentration of fructose 2,6-bisphosphate (which promotes glycolysis) and as such enhances rate of gluconeogenesis in the liver. Fructose 2,6-BP and AMP are increased in well-fed state and decreased in fasting state.

Third irreversible step bypassed during gluconeogenesis: Converting glucose-6-phosphate to glucose

This is done using enzyme glucose-6-phosphatase, which is only expressed in the liver and renal cortex. Insulin decreases glucose-6-phosphatase expression and glucagon increases its expression.

Acetyl CoA carboxylase (ACC): -how does ACC inhibit FA oxidation? -what activates it/inhibits it? -how does phosphorylation regulate it

This step inhibits FA oxidation thru inhibition of CPT-1 Malonyl CoA production is a rate limiting step; ACC is a key regulatory enzyme It is activated by citrate to produce active polymers, and deactivated by fatty acyl CoA (aka its product) Phosphorylation deactivates the enzyme in response to epinephrine (similar to Glucagon), cAMP, and protein kinase Dephosphorylation (due to insulin) activates the enzyme Basically inactive when attached to PO4- and active when not attached to it

FA synthesis requires lots of acetyl CoA and ATP

Transfer of acetyl CoA from mitochondria to cytosol involves the citrate shuttle Occurs when citrate concentration in mitochondria is high due to inhibition of isocitrate dehydrogenase by high levels of ATP. (Note: High ATP levels are also required for FA synthesis.)

How to create NADPH non-oxidatevly, or after forming ribulose-5-phosphate

Transketolase and transaldolase can convert ribulose-5-P into G3P or F6P, which can go into glycolysis

Why are triacylglycerols (TAG) better at storing energy?

Triacylglycerols stored in adipose tissue are our principal stored energy reserve Because its energy rich, inert, plays no functional role for the most part (open to being broken down), and very hydrophobic

T/F Double C=C bonds are introduced into fatty acids in the endoplasmic reticulum compartment of the cell.

True

T/F Saturated fatty acids contain zero alkene bonds

True

T/F The brain uses ketones instead of glucose for ATP production during long-term fasting.

True

T/F The enzyme that catalyzes the addition of an activated two-carbon compound with an activated three-carbon compound is β-ketoacyl synthase

True

T/F Untreated, chronic ketone body production will lower blood pH levels.

True

T/F When Ribose 5-phophate needs are greater than the need for NADPH, Glycolysis and the backwards route of the non-oxidative part of PPP are used.

True

T/F A biotin-deficient diet may lead to an increase of cytosolic acetyl CoA.

True

True or false? 1. Glycolysis produces a net 2 moles of ATP per 1 mole of glucose.

True

True or false? Acetyl CoA carboxylase is the enzyme that catalyzes the committed step in fatty acid synthesis.

True

True or false? Acetyl carboxylase is globally regulated by phosphorylation and dephosphorylation.

True

True or false? Glucagon stimulates gluconeogenesis.

True

True or false? In the phosphopentose isomerase reaction Ribulose 5-phosphate is converted to Ribose 5-phosphate through an enediolate intermediate

True

True or false? Reduced dihydrolipoamide is oxidized by transferring electrons and protons to NAD+.

True

True or false? ATP is called the energy currency whereas the currency of reductive power is NADPH.

True

True or false? Activation of the pyruvate dehydrogenase kinase decreases production of acetyl CoA.

True

True or false? Aldolase hydrolyzes Fructose 1,6 bisphosphate to produce glyceraldehyde 3-phosphate and dihydroxyacetone phosphate.

True

True or false? GAPDH (G3P dehydrogenase) uses NAD+ to oxidize glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate.

True

True or false? Phosphofructokinase-1 is the rate-limiting step of glycolysis.

True

True or false? The reaction Oxaloacetate → Phosphoenolpyruvate is unique to gluconeogenesis.

True

True/False The availability of the metabolite oxaloacetate determines whether acetyl CoA, made from fatty acids, can enter the citric acid cycle.

True

T/F Pyruvate needs to decarboxylated before acetyl-CoA can be formed.

True:

True or false? Lipoamide is covalently linked to E2 of pyruvate dehydrogenase through an amide bond to the side chain of a Lys residue.

True:

Describe how VLDL acts to transport triacylglycerol from the liver, and the role of Lipoprotein Lipase (LPL) in delivering FA's to tissues. How does insulin affect LPL activity in adipose tissue?

VLDL are very-low density lipoproteins that take the fatty acids synthesized in the liver from glucose, and transport them to various tissues and can end up in capillary beds. Lipoprotein lipase are located in capillary beds and work to take the TAG in the chylomicron (carrier molecules) and break them up into individual fatty acids. LPL also can also release the FA's in VLDL. LPL is activated by insulin.

Which vitamin gives rise to the cofactor in transketolase?

Vitamin B1 (thiamine)

First step in FA synthesis is synthesis of malonyl CoA:

We first have to get acetyl coA into the cytosol from the mitochondria using the citrate shuttle. Citrate then recombines with CoA and uses an ATP to re-create Acetyl CoA That acetyl CoA is then converted into malonyl CoA using the enzyme acetyl CoA carboxylase (as well as biotin, CO2, and ATP) This conversion of acetyl CoA to malonyl-CoA is the rate-limiting step

Oxidation and reduction reactions are always coupled

When pyruvate is transformed to lactate, it is reduced, and NADH gets oxidized (giving a negative delta G) The reverse is where lactate is oxidized, then in that case NAD+ is reduced to NADH

In which one of the following periods will fatty acids be the major source of fuel for the tissues of the body?

While running the last mile of a marathon

Type 1 diabetes and ketoacidosis:

Why do type I diabetics develop ketoacidosis*, but type II diabetics don't? In type I diabetics, there is no insulin, so, no malonyl CoA is produced, leading to unrestricted entry of FAs into the liver mitochondria and their conversion to ketones...

TCA Cycle Reaction 4: Oxidative-decarboxylation of a-ketoglutarate into succinyl-CoA

a-Ketoglutarate is converted into succinyl-CoA through a-Ketoglutarate dehydrogenase complex IRREVERSIBLE, negative delta G. Requires input of CoA-SH and NAD+ gets reduced to NADH (second NADH formed), 2nd CO2 released Coenzymes TPP, lipoic acid, FAD, NAD+ and CoA are required END OF STAGE 1

Which of the following is most likely to occur in a normal individual after ingesting a high carbohydrate meal? Only insulin levels decrease Only insulin levels increase Only glucagon levels increase Both insulin and glucagon levels decrease Both insulin and glucagon levels increase

b) only insulin levels increase

Which one of the following reactions is unique to gluconeogenesis? a) Lactate to pyruvate b) Phosphoenolpyruvate to pyruvate c) Oxaloacetate to phosphoenolpyruvate d) Glucose-6-PO4 to Fructose-6-PO4 e) 1,3-Bis-phosphoglycerate to 3-phosphoglycerate

c) Oxaloacetate to phosphoenolpyruvate

The synthesis of glucose from pyruvate by gluconeogenesis: a) occurs exclusively in the cytosol. b) is inhibited by an elevated level of glucagon. c) requires the participation of biotin. d) involves lactate as an intermediate. e) Requires the oxidation/reduction of FAD

c) requires the participation of biotin.

Phosphorylation of fructose: -fructokinase?

can either be phosphorylated by hexokinase (which prefers glucose) or fructokinase. Fructokinase is found in the liver, kidney and small bowel. It produces fructose-1-phosphate.

Warburg Effect

cancer cells use glycolysis at a much higher rate than normal tissue even when oxygen is available. Cells with mutant p53 (tumor suppressor protein) are defective in ETC and are forced to rely heavily on glycolysis for ATP production. This is achieved in part by increased synthesis of hexokinase and glucose transporters on plasma membrane It underlies the basis of PET scans (cancerous tumors uptake more glucose) Cancer of brain and bladders/kidneys can't be diagnosed by PET scan b/c they consume most glucose in body

Propionic acidemia

caused by proprionyl CoA carboxylase deficiency Presents in the early neonatal period with progressive encephalopathy and can also damage the heart, and liver, (very serious, treatment by strict dietary regime & biotin supplementation). Monitored by measuring propionic acid in the blood. Propionyl-CoA is also produced during the degradation of branched chained amino acids (isoleucine, valine, threonine, and methionine). In B12 deficiency (& mutase deficiency), methylmalonic acid is excreted in the urine.

Regulation of TCA Cycle:

citrate synthase is inhibited by its product citrate (no allosteric regulators). respiratory control (see next card) Ca2+ Regulation of isocitrate dehydrogenase

Von Gierke's Disease

glycogen storage disease (glucose-6-phosphatase deficiency) characterized by severe hypoglycemia, hepatomegaly, lipidemia, and lactic acidosis

GLUT 4

insulin-dependent glucose transporter in the muscle and adipose tissue that has high affinity for glucose (low Km); around 3 to 5 mM

Carbon substrates for gluconeogenesis (how to get pyruvate to start the pathway):

lactate, alanine, oxaloacetate (from TCA cycle), or jump right into converting G3P to DHAP with glycerol.

Which of the following would be expected to occur after acute alcohol ingestion? The activation of fatty acid oxidation. Lactic acidosis. The inhibition of ketogenesis. An increase in NAD/NADH+ ratio. An increase in gluconeogenesis.

lactic acidosis

Pyruvate carboxylase is activated by:

presence of acetyl CoA (from beta-oxidation due to increased glucagon)

Gluconeogenesis definition:

synthesis of glucose from molecules that are not carbs, such as precursors lactate, glycerol and amino acids (particularly alanine) to make pyruvate. primarily occurs in the liver (10% in the renal cortex, and around 40% in the renal cortex during starvation). It prevails in situations with decreased insulin, increased glucagon and increased fatty acids. It is essentially "reverse glycolysis" besides the 3 irreversible steps of glycolysis.

Role of Xylulose 5-phosphate in carbohydrate & fatty acid metabolism

• Excess carbohydrate (+Insulin) promotes lipid and TAG biosynthesis in the liver. • Glucose influx results in increased Xylulose-5-phosphate production. • Xylulose-5-phosphate activates protein phosphatase 2A (PP2A). • PP2A dephosphorylates and activates ChREBP (Carbohydrate Response Element Binding Protein), causing its nuclear translocation. • ChREBP increases the transcription of PK (glycolysis) and genes involved in fatty acid synthesis (malic enzyme (ME), ATP citrate lyase (ACL), acetyl-CoA carboxylase (ACC), fatty acid synthetase (FAS),and stearoyl-CoA desaturase-1(SCD1) ⇒ increased lipid synthesis