PHAR3316 Pharmacy Biochemistry
Exam 4: The super Cram
Chapter 4: The lipid bilayer
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Made of amphipathic molecules where the “heads” are exposed to the aqueous solution.
Triacylglycerol and fatty acids do not have the geometry to form a bilayer.
The center of the bilayer is extremely fluid.
Is a fluid asymmetrical structure.
The longer the chain or less saturated it is, the higher the melting point and less mobile it is.
Cholesterol in the lipid membrane can restrict lipid movement or can be used to prevent lipid
packing
Membrane rafts are unique structures consisting of packed cholesterol and sphingolipids that have
less fluidity.
Compared to lateral diffusion, transverse diffusion occurs at an extremely slow rate unless aided by
translocase/flippase.
The membrane consists of phospholipids and contains 50% protein by weight on average.
Can be integral, peripheral, or a lipid-linked protein
α helixes with hydrophobic side chains can easily cross bilayer.
In order for β strands to cross the membrane, eight strands must form a barrow shape.
Lipid-linked proteins are typically soluble.
Sphingolipids, glycoproteins, glycolipids, and lipid-linked proteins are found on the exterior portion
of the membrane.
Lipid-linked proteins and integral proteins do not diffuse easily due to interactions with other
proteins and the cytoskeleton.
Chapter 10: Signaling
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Signal transduction occurs when a ligand binds to a receptor and causes changes within the cell.
Agonist vs. Antagonist
Receptor binding is similar to enzyme binding, ie. Formula, hyperbolic graph
Most signaling occurs though G-protein coupled receptors and tyrosine kinase receptors.
A small signal can have large cellular effects through second messengers.
Second messengers can be nucleotides and their derivatives, polar and nonpolar lipids,
Speed, strength, and duration of signal depend on location, branching of pathway, and inhibitors.
G Protein a lipid-linked protein named for their ability to bind to GTP/GDP
Receptor portion is 7 α helixes in barrow like shape, main example β2-adrenergic receptor
Large variety of binding sites revealing specificity of ligands
The inactive G protein consists of a γ, β, and GDP bound to α subunit.
β2-adrenergic receptor binds to epinephrine and norepinephrine
1. Binding of receptor-ligand causes conformation changes and the interaction with the αsubunit of the G protein.
2. GDP is released and GTP is bound instead.
3. α-subunit dissociates from βγ subunit making both units active.
PHAR3316 Pharmacy Biochemistry
Exam 4: The super Cram
a. Activates adenylate cyclase (integral enzyme) which converts ATP into cAMP
i. cAMP is broken down by cAMP phosphodiesterase
b. cAMP binds and activates protein kinase A
i. transfers phosphate groups to ser/thr
ii. activation glycogen phosphorylase (breaks glycogen into glucose)
iii. self-regulated by phosphorylation
4. GTPase hydrolyze GTP returning to inactive state
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Desensitization by arrestin and ligand dissociation can also halt the pathway
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In α-adrenergic receptors, phospholipase C is activated converting phosphatidylinositol into inositol
trisphosphate and diacylglycerol. Inactivated by lipid phosphatases
1. Inositol triphosphate acts as a second messenger that triggers calcium channels in the ER
membrane to open.
2. Ca2+ ions activate protein kinase B
3. Can be phosphorylated more to produce different second messengers
4. Diacyglycerol is a second messenger that actives protein kinase C (responsible for cell
division and gene expression protein activation)
a. Uncontrolled activation of kinase C leads to tumor growth
b. Protein kinase C can also be activated by receptor tyrosine kinases
Receptor tyrosine kinase such as insulin undergo conformation changes in a way that the two tyrosine
kinase domains come together
1. Autophosphorylation occurs
2. Adapter proteins for a bridge activating G protein (GDP-> GTP)
3. G proteins activate a variety of kinases
Lipid Hormone signaling
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Hormone binding to receptors form dimers
The hormone-receptor complex bind to DNA binding domains consists of two zinc fingers and cross
linked 4 cys chains.
Eiconsanoids are produced in response and bind to GPCR. They degrade rapidly and are short
ranged.
Chapter 12: Metabolism
PHAR3316 Pharmacy Biochemistry
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Exam 4: The super Cram
The body breaks down polymers into monomer units for metabolism. The liver is responsible for
catabolizing, storing, and releasing the nutrients into the blood stream.
In order to obtain glucose from glycogen, it has to be phosphorylated.
Adipose tissue can store large amounts of triacylglycerols because they are hydrophobic.
The CNS requires glucose to function and the heart requires fatty acids for energy.
Metabolic pathways are interconnected and can be regulated by large free energy barriers.
Acetyl-coA can release lots of energy via thioester bonds.
Chapter 13: Glycolysis/Gluconeogenesis
Three key regulatory steps and large change in standard energy (know structure of substrate/product):
1. Hexokinase converts glucose to glucose -6 - phosphate
2. Phosphofructokinase converts fructose-6-phosphate into fructose-1,6-phosphate
3. Pyruvate kinase converts phosphoenolpyruvate into pyruvate
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During glycolysis, phosphorylation occurs at step 1 hexokinase, 3 phosphofructokinase, and 6
glyceraldehyde-3-phosphate dehydrogenase. Dephosphorylation occurs at step 7 phosphoglycerate
kinase and 10 pyruvate kinase.
Pyruvate can be converted to oxaloacetate for citric acid cycle or acetyl-coA for citric acid cycle/lipid
synthesis. It can be converted to lactate via Cori Cycle and into amino acids via α-ketoglutamate.
The conversion of pyruvate to lactate is important in the regeneration of NAD+ from NADH and
rapid production of short term ATP. IT is also a necessary step to regenerate NAD which is used in
the substrate level phosphorylation reaction of glycolysis. *step 6
Glycolysis occurs in the cells’ cytoplasm while gluconeogenesis occurs within the liver.
In gluconeogenesis, the hydrolysis of ATP (pyruvate carboxylase) and GTP (phosphoenolpyruvate
carboxykinase) is required to go from pyruvate to phosphoenolpyruvate due to the irreversibility of
the reaction in glycolysis. Note the large negative free energy barrier. The other energy barriers are
overcome by different enzymes that make the reaction favorable. (fructose biphosphatase and
glucose-6- phosphatase)
Fatty acids cannot be used for gluconeogenesis because they cannot be converted into
oxaloacetate.
Glucose-6-phosphate is converted ribulose-5-phosphate via pentose phosphate pathway (PPP). This
pathway generates two NADPH, two H+, and CO2.
PHAR3316 Pharmacy Biochemistry
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Exam 4: The super Cram
What are the key glycolytic cancer targets articulated in the paper you have read?
1. What are the metabolic characteristics of cancer cells?
High Metabolic rate, most of the pyruvate is directed toward anaerobic reactions – lactate for
energy.
A. Glycolysis
Hexose kinase:
Phosphofructose kinase:
Pyruvate Kinase: isoform is unregulated -> feed forward process
Lactate dehydrogenase: converts pyruvate to lactate
B. Gluconeogenesis: constant gluconeogenesis for upkeep of the cell. Lots of glutamate,
glutamine, α-ketoglutarate used up to convert nonessential amino acids into pyruvate.
C. Pentose Phosphate Pathway: NADPH and R-5-B is produced for nucleotide synthesis and cell
replication.
2. What mechanisms of cancer cells use to implement metabolic change?
Isoforms of three regulatory enzymes is the mechanism in which cancer cells implement
metabolic change.
3. What metabolites are essential for cancer cells and how do they obtain them?
Pyruvate, amino acids, and nucleotides are essential to cancer cells because of constant growth
and replication. The cells obtain them through glycolysis, transamination, and PPP.
4. What are direct and indirect mechanisms of inhibition of tumor metabolism (give examples?
Indirect targets include signaling pathways that are activated or repressed in tumors, resulting in
aberrant metabolic control, and direct targets consist of the metabolic enzymes themselves.
Examples of direct targeting are inhibiting nucleotide synthesis via PPP. Indirect targeting is
preventing glucose uptake via insulin pathway.
5. What are antimetabolites? Give examples in the area of cancer metabolism
In the manuscript there is a presentation on nucleotide biosynthesis and its inhibitors.
Background for this section is found in Chapter 18 section 18-3 pp: 480-481
6. What are isoforms? Give examples in the area of cancer metabolism
An isoform is an alternative form of the enzyme. An example is PKM1 vs. PKM2 in cancer cells.
Pyruvate kinase is an important regulated step however the isoform does not regulate the
pathway allowing for increased pyruvate production and use for tumor cells.
7. How is the glycolytic pathway used in cancer cells (what purpose)?
The glycolytic pathway is used to produce pyruvate for conversion to lactate for energy use.
8. How do different tissues utilize pathways differently?
The heart burns fatty acids and has plenty of mitochondria to carry out TCA/β-oxidation.
PHAR3316 Pharmacy Biochemistry
Exam 4: The super Cram
The liver uses ATP to produce glucose from lactate that arrives from the muscles, store glucose
as glycogen, and produce ketone bodies.
The muscles can store glycogen, consume pyruvate, and amino acids for energy.
Adipose tissue convert glucose into triacylglycerols.
The kidney can produce glucose via deamination of non-essential amino acids.
9. How do metabolic reactions in the mitochondria differ from those in the cytoplasm? How do
these two compartments communicate?
In the mitochondria, fatty acid degradation occurs and the citric acid cycle. In the cytoplasm,
glycolysis and fatty acid synthesis occurs.
Malonyl-CoA inhibits β-oxidation by inhibiting carnitine acytransferase, so that no fatty acids can
be delivered into the mitochondria.
10. List the different metabolic targets for cancer therapy and explain the selectivity of these for
cancer cells?
This is articulated in the article
11. Explain a feed forward process work in the glycolytic pathway of cancer cells?
The feed forward process involves pyruvate kinase. PKM2 is not inhibited by fructose-1,6phosphate allowing flux of pyruvate. PKM1 is inhibited by the molecule thus regulating the
glycolytic pathway.
12. What is the normal fate of pyruvate and how does that change in cancer cells?
The normal route of pyruvate is to the citric acid cycle. Instead the cells act under anaerobic
conditions and convert pyruvate into lactate for energy.
13. Why is pH regulation an important target in cancer (who?)?
pH regulation is a target in cancer because it inhibits glycolytic energy production. This is
because H+ ions allow lactate to be moved out of the cell. The surrounding cells can recycle
lactate to pyruvate for cancer cell use. Acidity allows the cells to become mobile, thus
neutralizing the pH prevents spread of the tumor.
14. What are some essential and non-essential amino acids?
Some amino acids are non-essential because they can be metabolized by the body
alanine
hydrophobic
arginine
charged
asparagine
polar
aspartate
charged
cysteine
polar
glutamate
charged
glutamine
polar
glycine
polar
proline
hydrophobic
serine
polar
tyrosine
polar
PHAR3316 Pharmacy Biochemistry
Exam 4: The super Cram
The amino acids that can’t be metabolized by the body are the essential amino acids.
histidine
polar
isoleucine
hydrophobic
leucine
hydrophobic
lysine
charged
methionine
hydrophobic
phenylalanine
hydrophobic
threonine
polar
tryptophan
hydrophobic
valine
hydrophobic
They are metabolized by transamination (amino group transfer) into acetyl-coA or
gluconeogenic precursors.
15. How does the amino acid metabolism tied to carbon metabolism and what is its importance in
cancer?
Amino acid metabolism is tied to carbon metabolism through a transaminase and α-keto acid,
example α-ketoglutarate. Through transaminase, amino acids can be converted into pyruvate,
oxaloacetate, or other citric acid precursors.
It is important in cancer cells because they have an extremely high metabolic rate compared to
other cells. Cancer cells obtain energy through the conversion of pyruvate to lactose due to lack
of vascularization and hostile environment due its metabolic rate. However this pathway only
yields two ATP thus they require a large source of pyruvate. In order to maintain its
proliferation, cancer cells must obtain pyruvate through the conversion of amino acids into
pyruvate. Glutamate, glutamine, and α-ketoglutamate are used to transfer amino groups in
order to produce pyruvate from non-essential amino acids. By targeting this pathway, cancer
cells do not have access to enough energy and essentially starve to death.
16. How are lipid biosynthesis and degradation tied to carbon metabolism? What compartments
do these activities take place?
Lipid degradation is tied to carbon metabolism because acetyl-CoA can be used for
gluconeogenesis and is one of the intermediates in the citric acid cycle. Lipid synthesis is tied to
carbon metabolism because it stores excess acetyl-CoA from the citric acid cycle for future use.
Lipid biosynthesis occurs in the cytosol and oxidation occurs in the mitochondria. Acetyl-CoA
can leave the mitochondria via conversion into citrate and then converted back to acetyl-CoA
for fatty acid synthesis. Fatty acids can be brought into the cell via transporter protein carnitine.
17. What is ketone? How are they synthesized? How are they used by different tissues? How is this
metabolism linked to amino acid, carbohydrate and lipid metabolism?
Ketone is a carbonyl with two side groups. Ketone bodies are produced from acetyl-CoA
by the liver for gluconeogenesis, example: acetoacetate and 3-hydroxybutyrate.
PHAR3316 Pharmacy Biochemistry
Exam 4: The super Cram
They are used by different tissues as metabolic fuel after being converted into acetyl-CoA.
Acetyl-coA goes into the citric acid cycle, first step in fatty acid synthesis. Ketone bodies
are water soluble so can be easily transported through the body without the use of
lipoproteins.
Ketogenesis occurs when there is a shortage of carbohydrates and is important in
reducing gluconeogenesis of amino acids. Acetyl-coA is a component of fatty acid and
cholesterol synthesis
Chapter 14: Citric acid cycle
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Decarboxylation of isocitrate and α-ketoglutate yields the two CO2 molecules.
The three regulated steps of the cycle are citrate synthase, isocitrate dehydrogenase, and αketoglutatarate dehydrogenase.
All the metabolites except for isocitrate and succinate are precursors for other pathways.
Citrate inhibits citrate synthase and phosphofructokinase.
Succinyl-CoA inhibits α-ketoglutate dehydrogenase and competitive inhibitor of citrate synthase.
NADH inhibits isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, and citrate synthase.
Ca+ ions activates isocitrate dehydrogenase and α-ketoglutatarate dehydrogenase.
ADP activates isocitrate dehydrogenase.
Increasing the concentration of its components set off a cascade of increased catalytic activity.
Pyruvate  Acetyl-CoA  citrate  isocitrate  α-ketoglutarate  succinyl –CoA  succinate …
Fumaratemalate oxaloacetateback to citrate
Chapter 17: Lipid metabolism
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Low density lipoproteins transport triacylglycerol to the body via VLDL. HDL transport excess
cholesterol back to liver.
β oxidation (4 steps) degrades acyl-CoA to produce acetyl-CoA, QH2, and NADH at the β carbon
(third carbon). It also sends electrons for oxidative phosphorylation.
Each round of oxidation degrades the acyl-CoA chain by two carbons and regulated by the
availability of CoA and NAD/NADH and Q/QH2 ratios.
Note that acetyl-CoA goes into the citric acid cycle in cases of low carbohydrate diet for a total of 14
ATP generated.
Fatty acid oxidation occurs in the mitochondria (primarily) and in peroxisomes.
Peroxisomes mainly oxidize long chains of fatty acid and called as such because the first step
transfers electrons to oxygen producing peroxisome. They are also used to oxidize branched fatty
acid strands.
Synthesis of fatty acids occurs in the cytosol up to 16 carbons and consumes 1 ATP per round.
Acetyl-CoA must bind with oxaloacetate in order to move in and out of the mitochondria which also
consume 1 ATP.
Fatty acids can be extended further through elongase.
The body can desaturate the fatty acid however essential fatty acids such as linoleate and linolenate
have to be obtained from diet.
PHAR3316 Pharmacy Biochemistry
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Exam 4: The super Cram
Ketone bodies (acetoacetate, 3-hydroxybutyrate) are synthesized from acetyl-CoA in the liver
mitochondria from fatty acid oxidation.
They are small and water –soluble and can easily be transported throughout the body without the
use of transporter proteins. It must be converted back to acetyl-CoA for use.
Cholesterol is synthesized in the cytosol and is also synthesized from acetyl-CoA.
The rate determining step in cholesterol synthesis is the conversion of HMG-CoA to mevalonate by
HMG-CoA reductase.
Chapter 19: Hormonal control
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Insulin stimulates glucose uptake via increased transporters on cell surface and inhibits glycogen
break down via inhibiting glycogen phosphorylase and activate glycogen synthase.
Insulin is receptor tyrosine kinase. Glucagon, epinephrine, and norepinephrine is GPCR.
Glucagon stimulates gluconeogenesis and inhibits storage.
Liver has glucokinase as oppose to hexokinase such as in other cells. The metabolism of glucokinase
stimulates insulin release.
Glucokinase has a high Km and has a sigmoidal curve.
Cells can use AMPK to regulate cell uptake of metabolites.
Adipose tissue can regulate appetite, insulin resistance, and fuel use through leptin, adiposenectin,
and resistin.
Chapter 20: DNA replication and repair
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DNA is replicated semi conservatively.
Writhe (supercoils) are energetically favorable and twisted negatively (will uncoil if stretched).
Topoisomerase is used to undo supercoils by cutting one (type I) or cutting both strands (type II).
DNA replication occurs through the “factory model of replication” where the replications complexes
are stationary/attached to plasma membrane.
Helicases unwinds the DNA for replication and is activated by phosphorylation by kinases.
Single-strand binding protein is important in preventing unwound DNA strands from reannealing
and nucleases.
Primase initiates the polymerization process by DNA polymerase by laying down the initial stretch of
RNA that will eventually be replaced.
DNA is synthesized in the 5’ to 3’ direction.
The “palm” portion of DNA polymerase has strong homology (conserved).
DNA has a second binding site that acts as an exonuclease when there are errors in replication.
(Changes in the open/closed conformation)
Nick translation –moving the nick in the 5’ to 3’ direction. DNA ligase Is needed join Okazaki
fragments.
Telomeres, telomerase, and POT1 combat the shortening of DNA due to lagging strand primers.
DNA damage can occur with mutations, mutagens, carcinogens, and oxidation
Transition mutation – purine to purine change
Transversion – purine to pyrimidine and vice versa
PHAR3316 Pharmacy Biochemistry
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Exam 4: The super Cram
Mismatched repair: MutS can locate mismatched nucleotides and create a kink that signals
endonucleases to cleave the strand upstream for repair.
Base excision repair: DNA glycosylases remove uracil base and recruits AP endonuclease that nicks
and creates a bend that signals for DNA polymerase.
Nucleotide excision repair: damaged nucleotides and the surrounding nucleotides is removed and
repaired by DNA polymerase.
Non homologous end joining –ku proteins bind to broken ends, recruits endonuclease, and using
polymerase to join strands
Recombination: using an undamaged strand as template to repair damaged strand. Recombination
genes function constitutively (always expressed).
Nucleosomes consist of 4 pairs of histone proteins for the core and one histone outside the core
that binds to DNA via hydrogen bonds and ionic interactions of the DNA backbone.
Modification of the histone tails open of the DNA for replication or transcription (acetylation).
DNA methyltransferases add methyl groups to C residues next to G residues. They are called CpG
islands and are located near the starting points of genes. Under normal circumstances, they are
unmethylated indicating that methylation silences the gene.
Polymerase chain reaction: A method developed to replicate DNA in vivo. It involves denaturing
DNA and then slowly allowing them to anneal in the presence of primase, free floating nucleotides,
and polymerase. This process is repeated to obtain a large amount of DNA