Download Biochemistry Final

CHEM345-Biochemistry
Final Exam
Fall 2007
HONOR CODE:
1. Using the some (does not have to be all) of the following vocabulary words, explain the
importance of integration in metabolism. Discuss common reaction themes. DO NOT DEFINE
EACH SEPARAETLY, rather define within your answer. This should be a summary of
metabolism. THIS SHOULD ONLY be 1-2 page of material. (30pts)
ATP
NADH
Acetyl CoA
Dehydrogenases
Phosphate
pyruvate
glycogen
glucose
CO2 and O2
glutamate
oxidation/reduction
phosphoryl transfer
fatty acids
compartmentalization
The overall goal of metabolism is to supply the body with energy, metabolites, and
macromolecules to carry out proper function and maintain homeostasis. Although carbohydrate
metabolism is the central pathway in all metabolism, the anabolic and catabolic pathways of
other macromolecules (lipids/fatty acids, proteins, and nucleic acids) follow the same guidelines
and have similar pathways to supply the body with what it needs. Metabolism is coordinated
based on the energy needs (ATP/ADP ratio) of the cells as well as the blood glucose level
(glucose, a carbohydrate, is the primary energy source of the body). It is imperative that blood
sugar be maintained to keep a constant supply of glucose to the brain, the largest consumer of
glucose in the body. Through enzymes called dehydrogenases, carbohydrates are oxidized to
release high energy electrons from their carbon-carbon bonds, reducing electron carriers (NAD+
→ NADH, FAD→FADH2), which carry electrons to the electron transport system to be used in
the production of ATP by oxidative phosphorylation when the cells are working in the low
energy state (low ATP/ADP ratio). Glucose is provided from the diet (circulating through the
blood) or glycogen stores in the liver, along with gluconeogenesis in the liver. When glucose
levels are low and glycogen stores have been depleted from the liver, and gluconeogenesis in the
liver is no longer effective in producing sufficient amounts of glucose, fatty acids and amino
acids are degraded to resupply glucose to maintain blood sugar (important for brain function) and
also to generate other intermediates that can be funneled into the TCA cycle to liberate high
energy electrons, but can come at higher costs to the body, primarily the brain and kidneys.
Although degradation of fatty acids can harbor lots of energy potential, under starving
conditions, degradation could be deadly. Under starving conditions, the brain is deprived of
glucose, and when glucose is scarce, fatty acids are converted via acetyl CoA to ketone bodies,
which temporarily replace glucose. When fatty acids are degraded in excess, these ketone bodies
create an acidic environment in the body, leading to acidosis and disrupting homeostasis, and
leading to organ malfunction. This is also true of excess amino acid degradation. When amino
acids are degraded, they release glutamate, which contains a carbon-carbon backbone to generate
glucose. To salvage this backbone, the amine group is released, creating urea. If too many
amino acids are degraded, this leads to increased amounts of urea, which is toxic to the body,
especially the kidneys. Under high ATP levels, the body does not need to produce excess ATP,
so oxidative pathways to release electrons are inhibited and glucose and other metabolites are
stored. Glucose can be reconverted from various metabolites that are in excess via
gluconeogenesis and excess glucose is stored in the liver as glycogen. If there are still excess
amounts of metabolites and metabolic intermediates, then they are stored as fatty acids and
lipids. For example, in the storage of fats, excess levels of acetyl CoA are converted to
cholesterol, a lipid molecule used to maintain membrane fluidity. Essentially, pyruvate, glucose6-phosphate, and acetyl CoA are the central molecules of metabolism because they are versatile
and can be funneled through many processes based on what is needed in the cell. Pyruvate is
probably the most flexible because it can be converted to enter numerous catabolic as well as
anabolic pathways, and can come from several different sources, as well as act as an allosteric
inhibitor. It is the product of glycolysis, which can be used to fuel the TCA cycle under low
ATP conditions by being converted to acetyl CoA; it can be reconverted to glucose in times of
high energy charge, and when ATP does not need to be made (primarily in the liver); it is a
primary molecule in the lactate salvage pathway. It is also a convergence point for other
macromolecular metabolic process, such as lipids/fatty acids and amino acid metabolism. In
fatty acid degradation, glycerol can be sent to the liver and converted to pyruvate via glycolysis,
which can it can be used to produce glucose by gluconeogenesis. During the degradation of
proteins, numerous amino acids can be degraded to pyruvate, either directly or indirectly. And
the reversal is true also – pyruvate can be degraded in order to help anabolize lipids and
macromolecules as well. Some of these aspects of pyruvate can be said for acetyl CoA as well.
Acetyl CoA can be produced readily from every macromolecule via degradation, but cannot be
reconverted quite as easily. Acetyl CoA cannot be used to regenerate glucose via
gluconeogenesis because it cannot be reconverted to pyruvate. So when acetyl CoA enters the
mitochondria, it is subject to numerous fates of either further catalysis or to fatty acid/lipid
synthesis, depending on what the cell needs and signals from the brain. Glucose-6-phosphate is
an important component of the pentose phosphate shunt, a series of side pathways that generates
glycolytic intermediates for glycolysis and the production of pyruvate, as well as intermediates
for other pathways, as well as ribose-5-phosphate (for the production of nucleotides). The
production and funneling of metabolic intermediates is one of the most important aspects of
metabolism. During degradation, intermediates from fatty acid and amino acid degradation can
be funneled into TCA so they can be utilized for ATP production, or these intermediates can be
funneled out of TCA in the production or storage of macromolecules. The fates of intermediates
are dependent on location (compartmentalization), energy charge of the cell, and blood sugar
level. If a particular intermediate is in one location with the cell being at a certain state, it is
subject to one type of reaction, while in another state, it may need to be funneled somewhere else
to perform another function.
2. Using figures in your as guides text, predict what would happen in 3 organ systems under
starvation (low sugar) conditions. How does Diabete mimic this situation. (HINT: remember
signaling and what’s on, what’s off and why?) 30pts
Under starvation conditions, blood sugar (blood glucose) levels are low and the energy charge of
the cell is low. The brain is the body’s largest consumer of glucose, and when deprived, it
begins to malfunction. Under these circumstances, the brain (particularly the adrenal medulla)
secretes glucagon in response to low glucose, which travels via the circulatory system to the liver
(which can be referred to as the “powerhouse” of metabolism because it performs numerous
metabolic activities) to degrade glycogen. Glucagon binds and facilitates a GPCR mediated
response, which stimulates a cAMP signal transduction cascade, activating protein kinase A and,
through a series of phosphorylations, ultimately activating glycogen phosphorylase a. This
enzyme catalyzes the cleavage of a single glucose molecule off of glycogen (via
phosphorylation) and glucose enters the blood, being delivered primarily to the brain to supply it
with the energy it needs, and some travels to the tissues as well to generate ATP necessary for
function. Gluconeogenesis is also on in the liver to generate glucose from free intermediates.
The liver can only store a day’s worth of glycogen, so when these stores get depleted, the brain is
once again deprived of the glucose it needs, so the body turns to fat degradation. The adipose
tissue houses triacylglycerols, which harbor great energy potential. In ultimate starvation, these
triacylglycerols are hydrolyzed to their glycerol and fatty acid components in the adipocytes.
Then, the glycerol can be funneled directly to the liver and converted to glucose via
gluconeogenesis, which then gets supplied to the brain by the blood. Fatty acids can be directed
to other cells, where they are converted to acetyl CoA and enter the TCA cycle to release
electrons and generate ATP. However, the glucose from glycerol may not be sufficient enough
to supply the brain with all the energy it needs, and as glucose levels keep getting smaller, the
brain is more and more deprived, and the fatty acids keep getting degraded. Fatty acids produce
acetyl CoA when oxidized, but acetyl CoA cannot be used to regenerate glucose (cannot enter
gluconeogenesis). Instead, in the liver (within the mitochondria of liver cells), acetyl CoA gets
converted to ketone bodies, which can be utilized by the brain in place of glucose for a short
while, but are insufficient. If acetyl CoA keeps being degraded into ketone bodies due to high
levels of fat degradation, there will be an increased level of ketone bodies circulating through the
blood and subsequently to the brain. Increased levels of these ketone bodies causes blood pH to
drop, which disrupts homeostasis and leads to metabolic and organ dysfunction. The brain is the
organ that is affected the most by the pH drop. Malfunction of the brain in ultimate starvation
results in death.
Diabetes is a reflection of this process because in diabetes, there is very little to no insulin
production, which is needed for the uptake of glucose from the blood. Instead, this leads to
increased levels of glucagon circulating through the system. Increased glucagon leads to
overstimulation of PKA and glycogen phosphorylase, which causes increased amounts of
glucose in circulation and increased glycogen depletion, with decreased amounts of uptake by
the cells, and less utilization of glucose for energy. The increased glucagon circulation leads the
body to believe that it is in starvation because glucose is not being taken up by the cells. Once
these stores are depleted, triacylglycerols are hydrolyzed at a faster rate, releasing increased
amounts of fatty acids and subsequently greater production of ketone bodies. Excess ketone
bodies leads to decreased pH in the blood, resulting in acidosis and subsequently diabetic coma
and death.
3. Amino acids contribute to biochemistry is multiple ways. Choose two global functions that
amino acids serve and describe in detail. (HINT: structure!!!) (20pts)
Amino acids are the building blocks of proteins. They constitute the primary sequence of
polypeptides, which are transcribed from genes in a DNA sequence into an mRNA strand.
Sequences of amino acids in a polypeptide determine the folding of secondary structures, or
structural motifs, known as alpha-helices and beta-pleated sheets. Secondary structures are
determined by the hydrogen bonds between individual amino acids of the peptide that come
together during folding. The R groups of individual amino acids are directed towards the outside
of the structure, giving it special characteristics, such as charge, that allow it to have special
interactions with the environment. For example, alpha-helices that have amino acids with
hydrophobic side groups tend to be found in hydrophobic environments, such as the plasma
membrane. These helices tend to be part of transmembrane proteins, where the transmembrane
domain is made of hydrophobic helices. These secondary structures are only determined by
pieces of the polypeptide based on sequence and structure of amino acids; in the end all of the
resultant secondary structures come together to form the final tertiary (3D) structure based on the
sequence of all the amino acids. This final structure is stabilized by noncovalent association
between neighboring amino acids in the 3D structure. Certain amino acids have come together
to form and stabilize the tertiary structure based on their electrochemical and physical properties.
The tertiary structure of the protein determines its function. Therefore, the primary sequence of
the polypeptide dictates the function of the protein. If there is an error in the primary sequence,
then the protein does not fold correctly and results in a faulty, inactive protein.
A major class of proteins is enzymes, which act as catalysts to speed up the rate of a spontaneous
chemical reaction. Enzymes contain active 3D clefts that bind a particular substrate (specific for
the enzyme), and catalyze a specific reaction (converting the substrate to a desired product). The
amino acids that are located within the catalytic site are the agents that help drive a particular
reaction forward; the shape and electrical environment of the catalytic site are dictated by the
amino acids. The arrangements of the amino acids (their location in the primary sequences allow
them to come together in the tertiary structure) as well as their electrical and chemical properties
(electrostatic interactions, hydrogen bonding capabilities, van der Waals and hydrophobic
interactions) contribute to the enzyme’s specificity for a particular substrate in a chemical
reaction. Interactions between the amino acids of the catalytic site (known as the catalytic
group) and the amino acids of the substrate allow for a perfect fit of the substrate into the site.
Therefore, substrate shape and shape of the active site should be complementary. The
electrochemical properties of the catalytic group facilitate the formation of the substrate’s
transition state and subsequently the final product. For example, serine proteases (such as
chymotrypsin) contain a “catalytic triad,” made up of a serine, a histidine, and an aspartate. The
chemical characteristics of the triad amino acids orient each other to create a strong nucleophile
in the catalytic site, which allows for sufficient cleavage of peptides via hydrolysis. The triad
dictates the shape of the catalytic site so the enzyme can only cleave peptide bonds on the
carbonyl side of amino acids that contain benzene rings, such as tryptophan and phenylalanine.
4. Eating a low carbohydrate/ high protein diet appears OK on the surface, but what happens if
you take it to the extreme. Using what you know about pathway regulation and on/off switches
explain what happens and why it can have a negative effect on the body overtime. (20pts)
Eating a low carbohydrate/high protein diet can seem okay because it results in quick weight
loss. But this loss is only short term, and prolonging this diet can have negative, even long term
effects on the body. Essentially, the body thinks it is in starving conditions because there is low
glucose (the primary energy source) circulating throughout, and less glucose is getting to the
brain. So the brain sends a signal, glucagon, to the liver to degrade glycogen in order to produce
glucose. Glygogen phosphorylase is activated from the protein kinase A signal transduction
cascade, which cleaves single glucose molecules from glycogen. Glycogen synthase has been
inactive by protein kinase A so as to not store glucose in the brain’s time of need.
Gluconeogenesis involving other metabolites is occurring in the liver as well. Glucose that
enters the muscles is entering glycolysis and cellular respiration to supply the muscles with
energy in order to perform optimally. Most of the glucose, however, travels to the brain. Once
glucose and glycogen stores have been depleted, the brain is still in constant need of energy, so
the body turns to fat degradation as well as amino acid degradation; amino acids are in
abundance because of increased levels of protein in the diet. Because fatty acids and amino
acids are not the primary energy molecules for the human body, increased catabolism of these
molecules can cause problem. The ingested protein gets digested to proteases in the small
intestine, and amino acids are released into the blood stream. These amino acids are utilized for
their carbon-carbon backbone to enter gluconeogenesis in the liver. In the liver, amino acids get
converted to glutamate. In order to be utilized for energy, glutamate must be stripped of its
amine group. This is done by glutamate dehydrogenase, in which glutamate gets oxidized to αketoglutarate and releasing an ammonium ion and NAD+ is reduced to NADH, which carries
electrons to be carried to the electron transport system. Under low energy charge, the αketoglutarate can be funneled into TCA in the muscles to liberate high energy electrons. Or αketoglutarate can be funneled via the malate-aspartate shuttle to the cytoplasm, where it can be
converted to oxaloacetate, a primary molecule in gluconeogenesis. However, this can only occur
in the liver because cells are actively metabolizing glucose through glycolysis. The ammonium
ion that is released is converted to urea via the urea cycle to leave the body because it is toxic. If
there are increased levels of protein degradation, urea levels are increasing. Because urea is
toxic, increased levels could have a serious impact on the body, primarily the kidneys. Because
the body cannot replenish degraded proteins, and proteins cannot be stored, the body will only
utilize enough amino acids so protein levels do not deplete. Thus the primary degradation
pathway in times of starvation is utilization of fats. Triacylglycols, the fat energy storage in the
adipose cells, are broken down into glycerol and fatty acids. Glycerol is transported via the
blood to the liver, where it is reconverted to glucose by gluconeogenesis to be supplied to the
brain, while fatty acids go through β-oxidation to produce acetyl CoA and can be funneled into
the TCA cycle to release electrons and produce ATP in the muscles. However, when fat
degradation is the primary metabolic pathway, there is decreased uptake of glucose by the
muscles from the blood because of high glucagon/low insulin levels, and fatty acids can diffuse
freely into the cell. There are increased levels of acetyl CoA in the cells from fatty acid
degradation; the increased acetyl CoA acts as an allosteric inhibitor of pyruvate dehydrogenase,
which stops the conversion of pyruvate to acetyl CoA. Thus, fatty acids become the major
energy source for muscle cells. Because of this increase in acetyl CoA, glycolysis is turned off,
and pyruvate can be sent to the liver to be converted to glucose by gluconeogenesis. The TCA
cycle cannot keep up with the increased influx of acetyl CoA, so excess amounts of acetyl CoA
are converted to ketone bodies in the liver and become the brain’s major source of fuel in the
absence of glucose. The brain can survive a shortThis is an inefficient way to supply the brain
with energy because increased levels of ketones circulating in the blood decreases the overall
pH, leading to acidosis and sickness, and possibly death.
6. Higher order organisms have evolved multi-subunited enzyme systems to control metabolic
pathways. Using one of the following a) describe the structure of the enzyme complex, b)
explain how it functions, c) explain how it is regulated, and d) explain why such enzyme
complexes arose. (20pts) You may use a diagram here.
Fatty Acid Synthatase
Pyruvate Dehydrogenase
Pyruvate dehydrogenase catalyzes the irreversible reaction of pyruvate → acetyl CoA. It is
irreversible because acetyl CoA cannot be converted back to pyruvate and subsequently glucose.
a. Pyruvate dehydrogenase is a large complex whose quaternary structure is composed of
three distinct enzymes
- Pyruvate dehydrogenase component (E1) – α2β2 tetramer
- Dihydrolipoyl transacetylase (E2) – catalytic trimer
- Dihydrolipoyl dehydrogenase (E3) – αβ dimer
The core of the complex is composed of 8 E2 enzymes (8 catalytic trimers) and each trimer has a
flexible lipoamide arm. 24 E1 and 12 E3 enzymes surround the active E2 core. This close
proximity increases the rate and efficiency of the reaction taking place and reduces the
possibility of unnecessary side reactions. This close proximity allows for movement of the
flexible lipoamide arm, which is a main component of the reaction process.
b. Pyruvate dehydrogenase acts by performing a decarboxylation, a series of
oxidation/reduction reactions involving cofactors, and transfer reactions in order to
decarboxylate pyruvate and convert it to acetyl CoA. Each subunit performs a particular
task in the overall reaction. E1, a dehydrogenase, performs an oxidative decarboxylation
of pyruvate, releasing CO2, and creating an active intermediate containing the desired
acetyl group with the use of the cofactor TPP. The lipoamide arm from E2 comes into
the E1 site and the acetyl group is transferred to the lipoamide arm, and then returns to
the active core. E2, a transacetylase, transfers the acetyl group to CoA, generating the
desired product. But the lipoamide arm needs to be reoxidized in order to carry out the
process again. So it swings into E3, a dehydrogenase, which gets oxidized by the FAD
cofactor (which in turn gets reduced, carrying electrons) and is ready to repeat the cycle.
The new FADH2 is then able to reduce an incoming NAD+ to generate NADH, which
can be used to carry electrons to the ETS, and FAD is reoxided so it too can take part in
the reaction once again.
c. Pyruvate dehydrogenase is regulated by multiple allosteric regulations and covalent
modification. Because it catalyzes an irreversible reaction, it is strongly subject to
regulation based on the needs of the cell, and can be inhibited or activated when
necessary. In times of high energy charge, (high ATP/ADP ratio) pyruvate
dehydrogenase can be allosterically regulated by the products that it helps to create –
acetyl CoA, ATP, and NADH. When these products are in abundance and do not need to
be produced any more, they will bind to an allosteric site on the enzyme and inhibit or
slow down the rate of the reaction it catalyzes because the cell does not need any more of
the molecule. Under low energy conditions, the enzyme needs to be activated to produce
acetyl CoA and subsequently ATP. It can be allosterically activated by ADP and
pyruvate and even CoA because they are in abundance and need to be converted to help
produce energy. Pyruvate dehydrogenase is also regulated by covalent modificiation. In
times when the products are in excess, then enzyme does not need to continue catalyzing
the reaction; phosphorylation of the E1 subunit by pyruvate dehydrogenase kinase
inactivates the enzyme and slows down the rate of the reaction. PDK can be activated by
the products of pyruvate dehydrogenase, which will then phosphorylate the enzyme to
slow it down. When there is a need for these products, then the dehydrogenase will be
dephosphorylated by its phosphatase, allowing it to produce product.
d.
7. Regulation is a key aspect of Biochemistry. We have seen this over and over again. Choose
one of the following topics and describe how “regulation” plays an important role. NOTE: We
are not just talking about enzymatic control, but others levels of control too. (20pts)
a. Protein Synthesis
b. Protein Folding
c. Compartmentalization
Compartmentalization, or location within the cell, is a major control mechanism because it
designates the fate of certain molecules and intermediates to specific metabolic pathways
depending on their location in the cell (the mitochondria or the cytoplasm) as well as the energy
needs of the cell. Molecules in one region of cell are subject to specific pathways than when
they are in another region. Therefore, the funneling of intermediates between the mitochondria
and the cytoplasm is tightly controlled. Under certain conditions, enzymes are allosterically
regulated and covalently modified in order to service the needs of the cell. In times of high
energy charge and the cell has certain metabolites in abundance, enzymes will be inactivated by
the products it produces (allosteric inhibition) or by phosphorylation (covalent modification).
Answer 1 (one) of the following:
8. You are a scientist at the New You Corporation. You are working hard to discover the protein
involved in “anti-aging”. You know only the amino acid sequence of this protein and the fact
that it binds to and catalyzes the addition of telomeres on the ends of genes (a process that halts
aging in cells).
a. In short answer suggest how you could purify this protein from a cell lysate. (10pts)
b. Upon purification you find you don’t have enough of the protein to use for your
assays. In short answer how could you make more of this protein? (10pts)
c. What assay could you perform to determine that this protein is fully functional and
ready to experimentation? (10pts)
9. Nothing is ever wasted in Biochemistry. Describe in detail two examples of this. Be sure to
explain why this is important. (30pts)
During periods of exercise and the cells are actively metabolizing, glucose gets converted to
pyruvate via glycolysis. During periods of intense exercise, pyruvate is being produced at excess
rates and oxygen is depleted quickly, creating an anaerobic environment. Instead of being
reduced to acetyl CoA by pyruvate dehydrogenase, pyruvate is reduced to lactate by lactate
dehydrogenase because pyruvate cannot enter the TCA cycle under anaerobic conditions.
Lactate cannot be further oxidized in the cells to yield energy; however, it can be recycled in the
liver to reproduce glucose. This process of salvaging lactate to regenerate glucose is called the
Cori Cycle, and shifts the weight of metabolism from the muscles to the liver. In order to be
metabolized further, lactate is channeled from the muscles to the liver by the circulatory system,
where lactate gets reduced back to pyruvate, which then gets converted back to glucose by
gluconeogenesis. This glucose is then funneled back to fuel the actively metabolizing tissues
and resupply glycolysis. Lactate salvage allows the body to maintain a balance of glucose in the
blood as well as the tissues. Glycolysis is responsible for making 4 ATP molecules; during this
intense exercise, it helps supply the muscles with small amounts of ATP that are sufficient for
quick bursts of activity. It also helps recycle NADH produced in glycolysis. When pyruvate is
reduced to lactate via lactate dehydrogenase, the enzyme also reoxidizes NADH into NAD+,
which is then funneled back into glycolysis in the oxidation of glucose to be used by other
dehydrogenases.
Even numbered fatty acids, the majority in the body, end up as two acetyl CoA molecules in the
last step of β-oxidation in the breakdown of fatty acids. However, there are some odd numbered
fatty acids as well, but they are not very common. They’re oxidation results in two different
molecules – one acetyl CoA (2 carbons) and one propionyl CoA (3 carbons). This propionyl
CoA does not have many functions by itself in the body, but because everything in biochemistry
is salvaged, it can be converted to another molecule and be useful somewhere else. Through a
carboxylation by propionyl CoA carboxylase, followed by an isomerization by a mutase,
propionyl CoA is converted to succinyl CoA, a major component of the TCA cycle. This
conversion is important because it allows for the utilization of a seemingly useless molecule,
which eventually gets oxidized in the TCA cycle to liberate electrons and produce ATP.
BONUS Question. 5pts.
What is the most beautiful thing you learned about Biochemistry this semester?