Exam 2 Pathways and enzymes

Question 1

GLUT transporters

Answer 1

• GLUT 1- in most tissues for basal glucose uptake
• GLUT 2- liver, intestine, and pancreas for high capacity glucose uptake (and glucose sensing in the pancreas). Independent of insulin, but insulin activates glycolysis and facilitates glucose utilization*
• GLUT 3- Brain for neuronal glucose uptake
• GLUT 4- muscle, adipose, and heart tissue for insulin-dependent glucose uptake

Question 2

Hexokinase regulation

Answer 2

Hexokinase is inhibited by G6P; preventing it from accumulating and tying up all of the cell’s store of inorganic phosphate

Question 3

Glucokinase regulation

Answer 3

Not inhibited by G6P like hexokinase. Glucokinase is regulated by its high Km and being bound to the glucokinase regulatory protein (GRP; promoted by F6P) in the fasted state; sequesters it in the nucleus. It is activated by higher glucose levels and by F1P mediated dissociation from GRP. Transcription in the liver is also induced by insulin (unlike hexokinase). When it is in the cytoplasm, glucokinase binds to the dephosphorylated form of F26BPase (form made from insulin activity; inactive form; b form) that keeps it in the active state (allows it to deal with the influx of glucose). Glucokinase is also inhibited by CoA thioesters and LCFAs (these are around in higher amounts in the liver when fasting occurs due to the livers consumption of them)

Question 4

phosphofructokinase-1 regulation

Answer 4

in liver: PFK1 is regulated by insulin/glucagon mediated F2,6BP (activator) from PFK2 and deactivated by citrate. Not under AMP regulation

In muscles/RBCs: ATP inhibits PFK1 and AMP activates PFK1

• Inhibited by ATP and stimulated by ADP and AMP in non hepatic tissues
• Inhibited by citrate in the liver (from the TCA cycle)
• Inhibited by low pH- prevent further accumulation of acidic byproducts when pH is already dangerously low
• Activated by F2,6BP in liver
• Stimulated by insulin and inhibited by glucagon (liver) or stimulated by insulin (muscle)

Question 5

Pyruvate kinase regulation

Answer 5

Activated by F1,6BP in all tissues and by ATP(-)/ADP/AMP(+) ratio in muscles and RBCs

In liver: regulated by phosphorylation from glucagon (inactivation) and feed forward activated by F1,6BP (not inhibited by ATP)

Question 6

glycolysis pathway

Answer 6

+hexokinase/glucokinase: glucose to G6P (uses ATP)
phosphoglucoisomerase: G6P to F6P
+PFK1: F6P to F1,6BP (uses ATP)
aldolase B: F1,6BP to DHAP and G3P
triose isomerase: DHAP to G3P
G3PDH: G3P to 1,3BPG (makes NADH and uses Pi)
Phosphoglycerate kinase: 1,3BPG to 3PG (makes ATP)
PG mutase: 3PG to 2PG
Enolase: 2PG to PEP (looses H2O)
+Pyruvate kinase: PEP to pyruvate (makes ATP)
+=irreversible

Question 7

gluconeogenesis pathway

Answer 7

+Pyruvate carboxylase (in mitochondria only): pyruvate to oxaloacetate
+PEPCK: oxaloacetate to PEP (uses GTP)
Back to F1,6BP through reversible reactions
+F1,6BPase: F1,6BP to F6P
Reversible phosphoglucoisomerase reaction
+G6Pase: G6P to glucose
+=irreversible

Question 8

bifunctional protein

Answer 8

has 2 domains; phophofructo-2-kinase and fructose-2,6-bisphosphatase. When the enzyme is phosphorylated by cAMP dependent PKA, it acts as a phosphatase and it acts as a kinase when it is dephosphorylated. F26BP stimulates phsophofructokinase and inhibits F16BPase, so when glucagon is active, the bifunctional protein is phosphorylated and acts as a phosphatase to decrease F26BP in the cell and allow gluconeogenesis

Question 9

fructose 1,6 bisphosphatase regulation

Answer 9

activated by: citrate and cortisol

deactivated by: AMP and F2,6BP

Question 10

Glycogen metabolism regulation

Answer 10

Glucagon: phosphorylation by PKA leads t phosphorylated , phosphorylase kinase, which phosphorylates glycogen phosphorylase (active), phosphorylated glycogen synthase (inactive), and phosphorylated inhibitor 1 protein (inhibits protein phosphatase 1; PP1). Epinephrine has the same effects in the liver and muscle; acts independently of dietary state. In muscle,

Insulin: leads to active PKB which causes exocytosis of GLUT4 transporters in some tissues, phosphorylated PP1 (active), which dephosphorylates glycogen synthase (active), glycogen phosphorylase, and phosphorylase kinase (inactive)

Allosteric regulation: G6P activates glycogen synthase and inactivates glycogen phosphorylase. Glucose and ATP also inhibit glycogen phosphorylase.

In muscle cells, phosphorylase kinase is also activated by Ca2+ and glycogen phosphorylase is activated by AMP without the need for other activators; allows the muscle to quickly adjust to demands before other regulation can catch up

Question 11

Ethanol Metabolism

Answer 11

• Converted to acetaldehyde tough alcohol dehydrogenase yielding NADH
• Acetaldehyde is converted to acetate in the mitochondria through acetaldehyde DH giving NADH
• This process gives a lot of NADH, so pyruvate is converted to lactate to give NAD+ through lactate DH, but in the fasted state, pyruvate is needed, so excessive drinking can lead to mild hypoglycemia because the accumulation of NADH in the mitochondria inhibits NADH shuttling to the mitochondria, leading to an increase in cytosolic NADH
• If too much NADH is accumulated, the second reaction involving the conversion of acetaldehyde is unable to proceed and acetaldehyde is responsible for the hangover through the formation of adducts with proteins and nucleic acids. Eventually resolves due to ETC use of NADH to regenerate NAD+
• NAD+ accumulation also inhibits gluconeogenesis because NAD+ is needed to oxidize lactate to pyruvate
• The final product of this, acetate, is converted to acetyl-coA and is used to synthesize fats. Chronic production of acetyl-coA leads to “fatty liver” seen in alcoholics
• When ethanol is present, there is a high amount of cytosolic and mitochondrial NADH, which ties up NAD+ and prevents conversion of lactate to pyruvate and malate to oxaloacetate which inhibits gluconeogenesis; not as much of a problem in adults as it is in children who have developing livers that are not yet efficient at this

Question 12

Fructose metabolism

Answer 12

• can be phosphorylated to F6P by hexokinase, but its Km for fructose is very high, so it is usually converted to F1P by fructokinase in the liver
• Aldolase B (same one that cleaves F1,6BP) then cleaves F1P into DHAP and glyceraldehyde
• Glyceraldehyde is converted to G3P by triode kinase using ATP to phosphorylate it.
• Excess fructose can lead to F1P accumulation due to fructose pathway bypassing rate limiting PFK1 step and aldolase B being slower than fructokinase due to it preferring F1,6BP as a substrate. F1P activates GRP, but has no affect on PFK, so stimulates glycogen synthesis, but also ties up a large amount of inorganic phosphate. The increased glycogen levels can also lead to shunting of glucose and fructose to lactic acid and FA production over time, possibly leading to accumulation of lipids in the liver if VLDLs can’t handle the FA load; this leads to FA liver disease
• Note; fructose cannot be converted to G6P, so it can’t enter the pentose phosphate pathway or glycolysis, so it is converted to fat when in excess in the liver. (In other tissues, hexokinase is used so it can be used like glucose, but other tissues only metabolize 10% of fructose).

Question 13

Galactose metabolism

Answer 13

• First metabolized by galactokinase which converts galactose to Gal1P using ATP. Irreversible
• Gal1P is then converted to UDP-Gal by Gal1P uridyl transferase using a UDP from a UDP glucose converting it to G1P. This reaction is reversible
• UDP-Gal is then converted to UDP-Glucose by UDP-galactose-4-epimerase. Reversible

Question 14

Pyruvate dehydrogenase information

Answer 14

• a multi enzyme complex with 3 components. These components channel the products to the next subunit, so it does not rely on diffusion of substrate.
  
  • Pyruvate dehydrogenase component (E1)- contains thaimine pyrophosphate as a prosthetic group (thyamine pyrophosphate is always involved in oxidative decarboxylations and most of the time involve in simple decarboxylations)
  • Dihydrolipoyl transacetylase component (E2)- contain lipoic acid on a lysine side chain
  • Dihydrolipoyl dehydrogenase component (E3)- contains FAD containing flavoprotein
  • There is a kinase that regulates this complex at E1 by phosphorylation. This enzyme is not regulated by PKA, but by ATP
  • NAD+ and CoA are also used as cosubstrates in the reaction
  • Except for lipoic acid, all of these cofactors require vitamins to be produced from the diet and deficiencies lead to pyruvate accumulation and subsequent reduction to lactate or transamination to alanine

Question 15

pyruvate dehydrogenase mechanism

Answer 15

• Pyruvate is decarboxylated and attached to thiamine pyrophosphate in E1 giving hydroxyethyl TPP
• The hydroxyethyl is released o the next enzyme E2 where the lypoyllysine is attached to this 2 carbon molecule
• Then acetyl coA is used in E2 to hydrolyze the molecule off, giving acetyl-CoA
• E3 is used to reoxidize the lipoyllysine using Fad to FADH2
• This FADH2 in E3 is oxidized by NADH which goes to the ETC and all of the cofactors are in their original states
• α-KGDH uses a similar mechanism
• PDH is inhibited by ATP, acetly-CoA, and NADH and is activated by AMP, CoA, and NAD+ and Ca+. The acetyl CoA regulation prevents accumulation of acetyl-CoA if there is not enough oxaloacetate to process it
• PDH is also covalently activated by dephosphorylation and inhibited by phosphorylation. This is done by a kinase that is activated by ATP

Question 16

TCA cycle

Answer 16

• Acetyl-coA is transferred to a 4 carbon oxaloacetate to form citrate through the enzyme citrate synthase. This reaction is irreversible
• Citrate is then isomerize to isocitrate by aconitase. It is first dehydrated to aconitate and then hydrated to isocitrate. This is reversible but the equilibrium is strongly to the citrate side
• Isocitrate is decarboxylated to α-ketoglutarate by isocitrate dehydrogenase. This is an irreversible step and produces a CO2 and NADH
• α-ketoglutarate is then converted to succinyl-CoA by α-ketoglutarate dehydrogenase; this enzyme resembles pyruvate dehydrogenase in structure and function. CO2 and NADH are also produced here.
• Succinyl-CoA synthetase (aka succinyl thiokinase) converts succinyl-Coa to succinate. There are 2 forms of this enzyme in humans; one gives ATP and one gives GTP. The one that gives ATP predominates in the brain and heart, the GTP generating one is in the liver. Most other tissues have both in similar amounts
• Succinate dehydrogenase complex (complex II) uses FAD to convert succinate in to fumarate, giving FADH2. This is embedded in the matrix and is used in the ETC
• Fumarate is then hydrated to malate through fumarase
• Malate is oxidized to oxaloacetate through malate dehydrogenase. Another molecule of NADH is produced here
• The TCA cycle yields 3 NADH and 1 FADH2 for each turn

Question 17

Pyruvate dehydrogenase regulation

Answer 17

• nhibited by NADH and acetyl-CoA, and is activated by NAD+, AMP, and CoA. It is also regulated by phosphorylation (inactivated). The protein that phosphorylates it is activated by the same things that inhibit pyruvate dehydrogenase (NADH and acetyl-CoA). They are also regulated by hormones, e.g. insulin inhibits pyruvate DH kinase, and allows pyruvate to oxidize pyruvate

Question 18

TCA regulation: Citrate synthase

Answer 18

inhibited by ATP and citrate (competes with oxaloacetate for the active site)

Question 19

TCA regulation: Isocitrate dehydrogenase

Answer 19

• nhibited by NADH and is activated by AMP and NAD+

Question 20

TCA regulation: α-ketoglutarate DH

Answer 20

inhibited by succinyl-CoA, ATP, and NADH

Question 21

Pasteur effect

Answer 21

flux of glucose through glycolysis is reduced under aerobic conditions (oxygen inhibit glucose consumption) due to the higher yield of glucose in the presence of oxygen. This effect is not seen in RBCs because they are strictly anaerobic

Question 22

Coenzyme Q (CoQ)

Answer 22

has an isoprene side chain that allows it to diffuse between the inner and outer leaflets of the inner membrane
All of the different pathways for elections converge on CoQ and then continue to the rest of the ETC

Question 23

Cytochrome C

Answer 23

• Cytochromes have red or brown heme proteins that are one-electron carriers. Except cytochrome C, they are all integral membrane proteins. Cytochrome C is water soluble and can escape, triggering apoptosis. These cytocromes are classified as a, b, or c depending on the spectral absorption wavelengths
• B and A cytohromes are noncolvaletnyl incorporated in the proteins, but C cytochrome are bound by cyctein

Question 24

Fructose

Answer 24

• Fructose is converted to F6P in muscles and adipose, but is converted to F1P in the liver, so will be unable to go anywhere except through FA synthesis because the gluconeogenic pathway is inactive when sugar is being taken up and glycolysis is active
• Fructose can deplete the liver of ATP/Pi due to overload of aldolase B, (this tying up of the inorganic phosphate can lead to inhibition of phosphorylysis of glycogen to glucose, leading to hypoglycemia- debated) so can lead to uric acid production because it tries to get inorganic phosphate thorough breakdown of nucleic acids, leading to hyperuricemia (gout); this can also be seen in chemotherapy, so needs to be treated with allopurinol with the chemo. Uric acid also inhibits NO synthase, leading to hypertension.

Question 25

mono-carboxylate transporter

Answer 25

• Lactate is transported to the mitochondria by the mono-carboxylate transporter and can be converted to pyruvate by irreversible isoform of lactate dehydrogenase. Pyruvate is also transported into the mitochondria by the mitochondrial pyruvate channel; but is quickly used by pyruvate dehydrogenase
• The mono-carboxylate transporter is also used to transfer lactate between astrocytes and neurons. The use of lactate allows neurons to bypass the glyoxylate cycle and prevent methylglyoxol formation; though it still does some classical glycolysis

Question 26

2,3BPG

Answer 26

RBCs use 2,3BPG to regulate Hb affinity for oxygen; 1,3BPG is converted to 2,3BPG by bisphosphoglycerate mutase and this is converted to 3 phosphoglycerate by bisphosphoglycerate kinase. This bypasses the phosphoglycerate kinase reaction that produces ATP, so looses 1 net ATP from glycolysis per molecules of 1,3BPG that goes through this

Question 27

Perilipin

Answer 27

protein that coats the lipid droplets in the adipose. It is phosphorylated by PKA and undergoes a conformational change that allows lipase access to the TAGs in the lipid droplet

Question 28

carnitine shuttle

Answer 28

• LCFAs are “activated” by adding acyl-CoA to them by ATP driven fatty-acyl coA synthetase; it then moves to the intermembrane space
• Carnitine palmitoyltransferase I (CPT 1) aka carnitine acyltransferase- transfers FA from FAcoA to carnitine, generating fatty acylcarnitine in the intermembrane space
  
  • Rate limiting enzyme of FA degradation
  • Inhibited by malonyl CoA (substrate of FA synthesis)
• Carnitine-acylcarnitine translocase (CACT)- anti porter that imports a FAcarnitine while exporting a carnitine molecule
• Carnitine palmitoyltransferase II (CPT II) transfers the FA from acylcarnitine to CoA in the matrix; β oxidation then begins

Question 29

Carnitine deficiency

Answer 29

can be due to inadequate diet (e.g. vegetarian- primary carnitine deficiency) or by genetic deficiencies. CPT II deficiency esp in cardiac and skeletal muscle can lead to cardiomyopathy and myoglobinuria after exercise. CPT I deficiency or CACT deficiency in the liver are rare and lead to death at a young age.

Question 30

β oxidation of Typical FAs

Answer 30

• Acyl-CoA DH- oxidized β-carbon producing FADH2 (electrons transferred to electron transferring flavoprotein (ETF), then to ETF:ubiquinone reductase, then to ubiquinone) and an enone called trans-enoyl-CoA
  
  • There are 4 different acyl-CoA DH enzymes for each of the FA lengths; SCAD, MCAD, LCAD, and VLCAD
  • MCAD deficiency is the most common deficiency with this step
• Enoyl-CoA hydratase- saturates the double bond with addition of water at β carbon to give β-hydroxyacyl CoA
• 3-hydroxyacyl CoA DH- oxidized β carbon to give β-ketoacyl CoA and NADH
• Acetyl CoA acyltransferase (aka β-ketothiolase)- attaches sulfur from CoA to ketone from other step generating acetyl-CoA and FACoA(n-2)

Question 31

β oxidation of Odd numbered FAs

Answer 31

undergo multiple round of β oxidation until they reach the 3 carbon propionyl CoA stage, then they are carboxylated to methylmalonyl CoA by propionyl CoA carboxylase using ATP (uses biotin cofactor). Methylmalonyl CoA mutase then isomerizes it to succinyl CoA (intermediate in TCA cycle). The enzyme uses cobamide (derivative of B12)

Question 32

β oxidation of Unsaturated FAs

Answer 32

undergo β oxidation until double bond is encountered, then enoyl CoA reductase reduces the double bonds that cannot be isomerized (2 double bonds at C’s 2 and 4) into a double bond at C3, and enoyl coA isomerase then converts the double bond at C3 into a neon (double bond at C2 adjacent to the ketone)

Question 33

VLCFAs β oxidation

Answer 33

• Oxidized in peroxisomes with some differences to the mitochondria:
• First step is catalyzed by a FAD containing acyl coA oxidase rather than a dehydrogenase, forming FADH2, which is used in the reduction of peroxide to water

Question 34

Glycerogenesis

Answer 34

as much as 40% of FAs used are recycled back into TAGs because accumulation of FAs is toxic so if release of FAs is too rapid, acts as a safety valve to counterbalance the excessive release of FAs

Question 35

Ketone body synthesis

Answer 35

• Acetyl CoA acetyltransferase- transfers an acetyl group from one acetyl CoA to the α carbon of another, generating acetoacetyl CoA
  
  • HMG CoA synthase transfers another acetyl group from another acetyl CoA to the β ketone of acetoacetyl CoA, generating HMG coA
    
    • HMG coA synthase is the rate limiting enzyme in ketone body synthesis
  • HMG CoA lyase- converts C3 hydroxyl group of HMG coA to β ketone by reduction, generating a proton, acetyl CoA, and acetoacetate.
  • Acetoacetate can spontaneously dexarboxylate to generate acetone which cannot be oxidized further, so it enters the blood stream and exits through the urine or breath (source of fruity smell in highly ketotic diabetics)
  • Acetoacetate can also be converted to β-hydroxybutyrate by 3-hydroxybutyrate DH which also oxidized NADH to NAD+

Question 36

Ketone body breakdown

Answer 36

• 3-hydroxybutyrate DH oxidized β-hydroxybutyrate back to acetoacetate yielding an NADH
• Acetoacetate CoA transferase- transfers CoA from succinyl CoA onto acetoacetate generating succinate and acetoacetyl CoA
  
  • This skips the succinyl CoA synthetase which would normally give a GTP in the TCA cycle
  • Liver cannot break down ketone bodies because liver cells lack succinyl CoA: acetoacetyl CoA transferase (aka thiophorase)
• Thiolase then converts acetoacetyl CoA into two molecules of acetyl CoA

Question 37

FA synthesis stages

Answer 37

• Occurs in 3 phases:
  
  • Entry of acetyl CoA into the cytosol by the citrate shuttle
  • Generation of malonyl coA by acetyl coA cargoxylase
  • FA chain formation where malonyl coA and acetyl coA are used to make FA chains in a repeating series of 7 reactions

Question 38

Entry of acetyl coA to the cytosol

Answer 38

• Acetyl coA must be in the cytosol to be used as a substrate for FA synthesis
• Uses the citrate shuttle to transport it out into the cytosol
  
  • Condenses acetyl coA with oxaloacetate into citrate by citrate synthase
  • Citrate is then transported into the cytosol by the citrate transporter
  • Acetyl coA is then regenerated from citrate through ATP citrate lyase which uses ATP and CoA to regenerate acetyl CoA and oxaloacetate
    
    • This enzyme is activated by glucose and insulin and inhibited by polyunsaturated fats and leptin
• Oxaloacetate regeneration by 2 possible mechanisms:
  
  • Cytosolic conversion of oxaloacetate to malate and importation of malate into the matrix by malate-α-ketoglutarate transporter which anti ports malate (in) and α-KG (out). Malate is then oxidized to oxaloacetate by malate DH (produces NADH)
  • Alternative: cytosolic malate is converted to pyruvate by the malic enzyme which uses NADP+ to NADPH. Pyruvate is then brought into the mitochondria where it is carboxylated by pyruvate carboxylase using ATP to form oxaloacetate (requires biotin)

Question 39

acetyl CoA carboxylase (ACC)

Answer 39

• Cytosolic Acetyl CoA is then converted to malonyl CoA by acetyl CoA carboxylase (ACC). This is the rate limiting enzyme of FA synthesis and uses ATP. The enzyme is active when dephosphorylated (insulin dependent) and present and a multiuser and deactivated when phosphorylated (glucagon, epinephrine dependent) and present as a dimer. It requires biotin and is allosterically activated by citrate and deactivated by AMP and palmitate. Genetic expression is also repressed by diets rich in polyunsaturated fatty acids (PUFAs)
  
  • Long term genetic expression is also regulated by diet: low fat/ high carb diet increases expression and high fat/fasting/glucagon decrease expression
  • Note: malonyl CoA prevents futile cycling by inhibiting carnitine acyltransferase I (CPT I), which inhibits β oxidation by inhibiting import of FACoA into the mitochondrial intermembrane space

Question 40

Synthesis of FAs

Answer 40

• Catalyzed by FA synthase (FAS)- large homodimer that catalyzes 6 reactions in 7 cycles followed by a 7th unique reaction
• FAS synthesis can be regulated by fasting/high fat or low carb diets (decreased expression) or high carb/low fat diets (higher expression)
• These are divided into 3 stages:
  
  • condensation
    
    • S-acetyl transferase activity of FAS- C2 of acetyl coA is transferred to cysteine on the acyl carrier protein (ACP) forming a thioester bond
    • S-malonyl transferase activity of FAS- C3 of malonyl coA is transferred to phosphopantheine residue of ACP (phosphopantheine is bound to ACP by serine)
    • 3-oxoacyl synthase activity of FAS- transfers acetyl group from cysteine to malonate and decarboxylates malonate forming 4 carbon β-ketoacyl group
  • reduction
    
    • FA synthesis uses NADPH from magic enzyme or from pentose phosphate pathway
    • 3-oxoacyl reductase activity of FAS- uses NADPH to reduce β-ketoacyl group to β-hydroxyl group
    • 3-hydroxypalmitoyl dehydrates- eliminates water from β-hydroxyl group to generate trans-none (double bond b/w C2/3)
    • Enoyl reductase- reduces the enone to a hydrocarbon using NADPH generating 4 carbon fattyacyl group attached to Pan-SH residue of FAS
• This cycle is repeated 6 more times; the 4 carbon fatty acyl group is transferred to the cysteine reside and an additional malonate group is bound to the Pan-SH group. This elongates the chain to 16Cs
• Third stage is product release; done by acyl hydrolase (aka thioesterase) activity of FAS which uses water to cleave off palmitate from the Pan-SH residue

Question 41

• Purine synthesis

Answer 41

• Dietary purines and pyrimidines are digested in the intestine. Pyrimidines are absorbed to a limited extent and purines are degraded to ribose and uric acid, so synthesis is critical
• Most de novo synthesis of purines occurs in the liver:
  
  • Begins with ribose-5-phosphate from the pentose phosphate pathway
  • 5-phosphoribosyl-1-pyrophosphate synthetase (PRPP synthetase) first forms PRPP by transferring pyrophosphate from ATP to C1 of ribose 5 phosphate (PRPP is activated form of ribose for nucleotide synthesis)
  • PRPP amidotransferase next replaces pyrophosphate group of PRPP with nitrogen from side chain of glutamine to form phosphoribosylamine. First committed step of purine biosynthesis
  • This is followed by a series of reactions that form the purine ring from several different building blocks using 4 ATPs. The first nucleotide formed in the pathway is inosine monophosphate (IMP) which contains the base hypoxanthine. IMP is a branch point between the synthesis of AMP and GMP
    
    • IMP is converted to adenylosuccinate by adenylosuccinate synthetase using Asp and GTP. Adenylosuccinate is then converted to AMP by adenylosuccinase which releases a fumarate from the adenylosuccinate
    • IMP is converted to xanthine monophosphate by IMP DH generating NADH. Xanthine monophosphate is then converted to GMP by GMP synthetase using glutamine to glutamate and ATP to AMP
  • The formation of purines is regulated by feedback inhibition: PRPP synthetase and PRPP amidotransferase are both inhibited by purine nucleotides. The reactions leading to IMP, AMP, and GMP are also inhibited by their end products

Question 42

• Purine degradation

Answer 42

• Degradation begins with hydrolysis of phosphates to nucleosides
• Nucleosides are then cleaved into ribose 1 phosphate and free base by purine nucleoside phosphorylase (adenine is a poor substrate for this, so it is deaminated to inosine first)
• Uric acid is the final product of purine degradation; synthesized by xanthine oxidase (sed for both steps) through xanthine and hypoxanthine. This enzyme contains FAD, a nonheme iron, and molybdenum. FAD is regenerated by transferring the H’s from FADH2 to oxygen forming peroxide. Uric acid is sythesized mainly in the liver. ~70% is excreted by the kidneys and ~30% by the intestine.

Question 43

• Purine salvage

Answer 43

• Free nucleotides can be recycled back into the nucleotide pool instead of being degraded into uric acid
  
  • Uses hypoxanthine-guanine phosphoribosyl transferase (HPRT) and adenine phosphoribosyl transferase (APRT)
  • ~90% of purine bases are recycled and many tissues can only get nucleotides from obtaining nucleotide bases in the bloodstream
  • HRPT is competitively inhibited by IMP an GMP and ARPT is inhibited by AMP

Question 44

Pyrimidine metabolism

Answer 44

• Pyrimidine ring is synthesized before the ribose is added unlike purine synthesis
• First, carbamoyl phosphate synthetase synthesizes carbamoyl phosphate from CO2 and glutamine. CPS I is present in the mitochondria and is used in the urea cycle while CPS II is in the cytosol and is used in de novo pyrimidine synthesis. CPS I uses ammonia from urea cycle as source of nitrogen rather than glutamine
• First pyrimidine is formed from carbamoyl phosphate, and aspartate, forms orotic acid which is processed to uridine nucleotides. The carbamoyl phosphate is synthesized by cytoplasmic carbamoyl phosphate synthetase II (uses nitrogen from glutamine instead of free ammonia like mitochondrial isozyme)
• The first 3 enzymes are carbamoyl phosphate synthetase II, aspartate transcarbamoylase, and dihydroorotate dehydrogenase are together in a single large polypeptide that is regulated by CTP as is CTP synthetase
• Dihydroorotate dehydrogenase is inhibited by leflunomide which blocks pyrimidine biosynthesis; used as immunomodulatory drug in rheumatoid and psoriatic arthritis
• Pyrimidines are degraded to water soluble products β-alanine or β-aminoisobutyrate

Question 45

deoxyribonucleotide syntesis

Answer 45

• Ribonucleotide reductase which reduces the ribose to deoxyribose forming NADPH. Levels of this enzyme rise immediately preceding S phase of the cell cycle. Also regulated allosterically:
  
  • ATP activates the enzyme and dATP is negative effector of all reactions. Other nucleotides regulate the specificity to guarantee balanced proportion of the 4 deoxydribonucleotides
• Thymine is synthesized from thymidylate synthase where the methylene group of THF is reduced to methyl group during transfer to dUMP and THF is oxidized to dihydrofolate. THF is regenerated from dihydrofolate by dihydrofolate reductase

Question 46

• Ubiquitin proteasome degradation

Answer 46

• Ub is a small 76 AA tag that is added to ε amino group of Lys on substrate
• Proteins are tagged by ubiquitin using 3 enzymes (E1, E2, and E3)
  
  • E1- Ub activating enzyme which binds Ub in the presence of ATP
  • E2- Ub is transferred to E2 (Ub carrier) from E1
  • E3- Ub ligase attaches the Ub onto the target protein
  • Subsequent Ub’s are added to the first
  • Usually at least 4-5 Ub’s are added to target a protein for degradation (lower # of Ubs can target a protein for transport to another compartment)
• Proteins with this Ub chain are targeted to the proteasome
  
  • The proteasome is a 26S complex made of 2 smaller subunits
  • 19S regulatory subunit recognizes the substrate and unfolds the peptide while removing the Ub’s to be recycled
  • 20S core unit degrades the protein in to mono, di, tri, or oligopeptides that can be used for amino acids or directed to antigen presentation

Question 47

Transamination

Answer 47

• α-amino group of amino acid is transferred to α-ketoglutarate to produce α ketoacids and glutamate; these reactions are reversible and use pyridoxal phosphate (PLP) cofactor. AKA transaminases or aminotransferases. Present ion the cytoplasm and mitochondria, esp in the liver, kidney, muscle, and intestine. All AAs except Lys, Pro, and The go through transamination in catabolism
  
  • All aminotransferases require PLP cofactor attached to ε amino group of lysine
  • Transfers amino group from AA to PLP forming pyradoxamine phosphate which can react with α kept acid to reform AA because Keq is close to 1. Allows creation to run in both directions depending on nutritional state
    
    • Alanine aminotransferase and aspartate aminotransferase are two most important because they are used leading up to formation of aspartate which enters the urea cycle
  • Glutamate DH reversibly uses NAD+ to NADH to release the ammonia from glutamate and form α-ketoglutarate. Reverse reaction uses NADPH and free ammonia. Glutamate is the only amino acid that undergoes oxidative deamination
  • Most amino acid nitrogen release forms glutamine or alanine rather than free ammonia
    
    • Alanine can enter the glucose-alanine cycle in the muscle where alanine is converted to pyruvate which can form glucose which can form alanine
    • Glutamine synthesis occurs in muscle and liver, but also important in nervous system
    • Ammonia can also be used to form urea in the liver; urea can be released into blood and excreted through kidneys or small intestine where bacteria metabolize it to ammonium and carbon dioxide; the ammonia can be reabsorbed as described above. Increased blood urea is uremia and is common in renal failure

Question 48

• Urea cycle

Answer 48

• Carbamoyl phosphate synthetase I uses 2 ATP to combine bicarbonate and ammonium to carbamoyl phosphate
• Ornithine transcarbamoylase transfers adds the carbamoyl from carbamoyl phosphate to ornithine forming citruline and a free phosphate.
• These first 2 reactions occur in the mitochondria and citruline is exported to the cytosol.
• Arginosuccinate synthetase combines citruline with aspartate using ATP to AMP to form arginosuccinate
• Argniosuccinate lyase releases fumarate and forms arginine
• Arginase hydrolyzes urea from arginine, forming ornithine which is imported back into the mitochondria
• Overall: 3 ATP, 1 Asp, 1 ammonia, and 1 CO2 are used; this cycle occurs only in the liver, so levels of argninase are only high in the liver
• Deficiency in transcarbamoylase results in hyperammoniemia and high levels of urine orotate (nucleic acid) because the equilibrium moves that direction
• CPS I catabolyzes rate limiting step and requires N-acetylglutamate for activity. Arginine stimulates the synthesis of N-acetylglutamate from glutamate which can be hydrolyzed to acetate and glutamate
• Enzymes that catalyze the first 4 reactions are found in liver, kidney, and intestinal mucosa, but only arginase is found in liver. The enzymes in the kidneys and intestine are used in biosynthesis of proteins
• The resulting fumarate can enter the TCA cycle or be converted to malate which can enter the TCA cycle