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chapter 23 protein turnover

Terms in this set (64)

Proteins are denatured by acid in the stomach. This
denaturation makes them better substrates for proteolysis. Explain
why this is the case.
When the proteins are denatured, all of the peptide bonds are accessible to proteolytic enzymes. If the three-dimensional structure of a protein is maintained, access to many peptide bonds is denied to the proteolytic enzymes.
What are the steps required to attach
ubiquitin to a target protein?
First, the ubiquitin-activating enzyme (E1) links ubiquitin to a sulfhydryl group on E1 itself. Next, the ubiquitin is transferred to a cysteine residue on the ubiquitin-conjugating enzyme (E2) by E2. The ubiquitin-protein ligase (E3), using the ubiquitinated E2 as a substrate, transfers the ubiquitin to the target protein.
a. Pepsin
7. Stomach proteolytic enzyme
b. N-terminal rule
4- Determines half-life of a
protein
c. Ubiquitin
2. Marks a protein for
destruction
d. PEST sequence
10. Pro-Glu-Ser-Thr
e. Threonine nucleophiles
5. 20S core
f. ATP-dependent protein unfolding
3. 19S regulatory subunit
g. Proteasome
9. Protein-degrading machine
h. Ubiquitin-activating enzyme
1. Requires an adenylate
intermediate
i. Ubiquitin-conjugating enzyme
6. Substrate for ligase
j. Ubiquitin-ligase
8. Recognizes protein to be
degraded
Protein hydrolysis is an exergonic process, yet the
26S proteasome is dependent on ATP hydrolysis for activity

a. Explain why ATP hydrolysis is required by the 26S proteasome.
b. Small peptides can be hydrolyzed without the expenditure of ATP.
How does this information concur with your answer to part a?
a. The ATPase activity of the 26S proteasome resides in the 19S subunit. The energy of ATP hydrolysis is used to unfold the substrate, which is too large to enter the catalytic barrel. ATP may also be required for translocation of the substrate into the barrel.

b. Substantiates the answer in part a. Because they are small, the peptides do not need to be unfolded. Moreover, small peptides could probably enter all at once and not require translocation.
The archaeal proteasome contains 14
identical active β subunits, whereas the eukaryotic proteasome has
two sets of 7 distinct β subunits. What are the potential benefits of
having several distinct active subunits?
In the eukaryotic proteasome, the distinct β subunits have different substrate specificities, allowing proteins to be more thoroughly degraded.
The 19S subunit of the proteasome contains six
subunits that are members of the AAA ATPase family. Other members
of this large family are associated into homohexamers with sixfold
symmetry. Propose a structure for the AAA ATPases within the 19S
proteasome. How might you test and refine your prediction?
The six subunits probably exist as a heterohexamer. Cross-linking experiments could test the model and help determine which subunits are adjacent to one another.
Aminotransferases require which of the following
cofactors:
a. NAD+/NADP+
b. Pyridoxal phosphate
c. Thiamine pyrophosphate
d. Biopterin
B
Butch Cassidy and the Sundance Kid. Glutamate dehydrogenase
requires which of the following cofactors:
a. NAD+/NADP+
b. Pyridoxal phosphate
c. Thiamine pyrophosphate
d. Biopterin
A
Which of the following compounds readily accepts amino
groups from amino acids?
a. Glutamine
b. Isocitrate
c. Malate
d. α-Ketoglutarate
D
The immediate donors of the nitrogen atoms of
urea are:
a. Aspartate and glutamate
b. Glutamate and carbamoyl phosphate
c. Aspartate and carbamoyl phosphate
d. Glutamine and aspartate
C
Which compound of the urea cycle is synthesized in the
mitochondria and transported into the cytoplasm?
a. Ornithine
b. Citrulline
c. Arginiosuccinate
d. Arginine
B
Keto counterparts. Name the α-ketoacid that is formed by
the transamination of each of the following amino acids: ,
a. Alanine
b. Leucine
c. Aspartate
d. Phenylalanine
e. Glutamate
f. Tyrosine
a. Pyruvate;
b. oxaloacetate;
c. α-ketoglutarate.
d. α-ketoisocaproate.
e. phenylpyruvate;
f. hydroxyphenylpyruvate.
A versatile building block.
a. Write a balanced equation for the conversion of aspartate into
glucose through the intermediate oxaloacetate. Which coenzymes
participate in this transformation?
b. Write a balanced equation for the conversion of aspartate into
oxaloacetate through the intermediate fumarate.
a. Aspartate+αketoglutarate+GTP+ATP+2 H2O+NADH+H+→1/2glucose+glutamate+CO The required coenzymes are pyridoxal phosphate in the transamination reaction and NAD+/NADH in the redox reactions.

Aspartate+CO2+NH4 ++3 ATP+NAD++4 H2O → oxaloacetate+urea+2 AD
Pyridoxal phosphate stabilizes carbanionic
intermediates by serving as an electron sink. Which other prosthetic
group catalyzes reactions in this way?
Thiamine pyrophosphate.
How do aminotransferases and glutamate
dehydrogenase cooperate in the metabolism of the amino group of
amino acids?
Aminotransferases transfer the α-amino group to α-ketoglutarate to form glutamate. Glutamate is oxidatively deaminated to form an ammonium ion.
What amino acids yield citric acid cycle
components and glycolysis intermediates when deaminated?
Aspartate (oxaloacetate), glutamate (α-ketoglutarate), alanine (pyruvate)
What amino acids can be deaminated directly
Serine and threonine.
What are the common features of the breakdown
products of the carbon skeletons of amino acids?
They are either fuels for the citric acid cycle, components of the citric acid cycle, or molecules that can be converted into a fuel for the citric acid cycle in one step.
Propose a role for the positively charged guanidinium
nitrogen atom in the cleavage of argininosuccinate into arginine and
fumarate.
It acts as an electron sink.
What are the immediate biochemical sources for
the two nitrogen atoms in urea?
Carbamoyl phosphate and aspartate.
a. Formed from NH4+
4. Carbamoyl phosphate
b. Hydrolyzed to yield urea
5. Arginine
c. A second source of nitrogen
1. Aspartate
d. Reacts with aspartate
6. Citrulline
e. Cleavage yields fumarate
7. Arginosuccinate
f. Accepts the first nitrogen
3. Ornithine
g. Final product
2. Urea
Identify structures A-D, and place them in the order that they
appear in the urea cycle.
A, arginine; B, citrulline; C, ornithine; D, arginosuccinate. The order of appearance: C, B, D, A.
Four high-transfer-potential phosphoryl groups
are consumed in the synthesis of urea according to the stoichiometry
given on page 766. In this reaction, aspartate is converted into
fumarate. Suppose that fumarate is converted into oxaloacetate.
What is the resulting stoichiometry of urea synthesis? How many
high-transfer-potential phosphoryl groups are spent?
CO2+NH4++3 ATP+NAD++aspartate+3 H2O→urea+2 ADP+2 Pi+AMP+PP Four high-transfer-potential phosphoryl groups are spent. Note, however, that an NADH is generated if fumarate is converted into oxaloacetate. NADH can generate 2.5 ATP in the electron-transport chain. Taking these ATP into account, only 1.5 high-transferpotential phosphoryl groups are spent.
A friend bets you a bazillion dollars that you can't prove
that the urea cycle is linked to the citric acid cycle and other
metabolic pathways. Can you collect?
The synthesis of fumarate by the urea cycle is important because it links the urea cycle and the citric acid cycle. Fumarate is hydrated to malate, which, in turn, is oxidized to oxaloacetate. Oxaloacetate has several possible fates: (1) transamination to aspartate, (2) conversion into glucose by the gluconeogenic pathway, (3) condensation with acetyl CoA to form citrate, or (4) conversion into pyruvate. You can collect.
Compound A has been synthesized as a potential
inhibitor of a urea-cycle enzyme. Which enzyme do you think
compound A might inhibit? Explain your answer.
Ornithine transcarbamoylase (Compound A is analogous to PALA
Glutamate is an important neurotransmitter whose
levels must be carefully regulated in the brain. Explain how a high
concentration of ammonia might disrupt this regulation. How might
a high concentration of ammonia alter the citric acid cycle?
Ammonia could lead to the amination of α-ketoglutarate, producing
a high concentration of glutamate in an unregulated fashion. αKetoglutarate for glutamate synthesis could be removed from the citric acid cycle, thereby diminishing the cell's respiration capacity
The urine of an infant gives a positive reaction
with 2,4-dinitrophenylhydrazine. Mass spectrometry shows
abnormally high blood levels of pyruvate, α-ketoglutarate,
and the α-ketoacids of valine, isoleucine,
leucine and threonine. Identify a likely molecular defect and propose
a definitive test of your diagnosis.
The mass spectrometric analysis strongly suggests that three enzymes—pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, and the branched-chain α-ketoacid dehydrogenase—are deficient. Most likely, the common E component of these enzymes is missing or defective. This proposal could be tested by purifying these three enzymes and assaying their ability to catalyze the regeneration of lipoamide.
How would you treat an infant who is deficient in
argininosuccinate synthetase? Which molecules would carry nitrogen
out of the body?
Benzoate, phenylacetate, and arginine would be given to supplement a protein-restricted diet. Nitrogen would emerge in hippurate, phenylacetylglutamine, and citrulline.
As we will see later (Chapter 27), liver damage
(cirrhosis) often results in ammonia poisoning. Explain why this is the
case.
The liver is the primary tissue for capturing nitrogen as urea. If the liver is damaged (for instance, by hepatitis or the excessive consumption of alcohol), free ammonia is released into the blood.
Argininosuccinic aciduria is a condition
that results when the urea-cycle enzyme argininosuccinase is
deficient. Argininosuccinate is present in the blood and urine.
Suggest how this condition might be treated while still removing
nitrogen from the body.
This defect can be partly bypassed by providing a surplus of arginine in the diet and restricting the total protein intake. In the liver, arginine is split into urea and ornithine, which then reacts with carbamoyl phosphate to form citrulline. This urea-cycle intermediate condenses with aspartate to yield argininosuccinate, which is then excreted. Note that two nitrogen atoms—one from carbamoyl phosphate and the other from aspartate—are eliminated from the body per molecule of arginine provided in the diet. In essence, argininosuccinate substitutes for urea in carrying nitrogen out of the body. The formation of argininosuccinate removes the nitrogen, and the restriction on protein intake relieves the aciduria.
Why should phenylketonurics avoid using aspartame, an artificial sweetener? (Hint: Aspartame is L-aspartyl-Lphenylalanine
methyl ester.)
Aspartame, a dipeptide ester (L-aspartyl-L-phenylalanine methyl ester), is hydrolyzed to L-aspartate and L-phenylalanine. High levels 3 of phenylalanine are harmful in phenylketonurics.
N-Acetylglutamate is required as a cofactor in the synthesis
of carbamoyl phosphate. How is N-acetylglutamate synthesized from
glutamate?
N-Acetylglutamate is synthesized from acetyl CoA and glutamate. Once again, acetyl CoA serves as an activated acetyl donor. This reaction is catalyzed by N-acetylglutamate synthase.
A deficiency of even one amino acid
results in a negative nitrogen balance. In this state, more protein is
degraded than is synthesized, and so more nitrogen is excreted than
is ingested. Why would protein be degraded if one amino acid were
missing?
Not all proteins are created equal: some are more important than others. Some proteins would be degraded to provide the missing amino acid. The nitrogen from the other amino acids would be excreted as urea. Consequently, more nitrogen would be excreted than ingested.
Differentiate between ketogenic amino acids and
glucogenic amino acids.
The carbon skeletons of ketogenic amino acids can be converted into ketone bodies or fatty acids. Only leucine and lysine are purely ketogenic. Glucogenic amino acids are those whose carbon skeletons can be converted into glucose.
The end products of tryptophan degradation are
acetyl CoA and acetoacetyl CoA, yet tryptophan is a gluconeogenic
amino acid in animals. Explain.
As shown in Figure 23.28, alanine, a gluconeogenic amino acid, is released during the metabolism of tryptophan to acetyl CoA and acetoacetyl CoA.
Pyruvate dehydrogenase complex and α-ketoglutarate
dehydrogenase complex are huge enzymes
consisting of three discrete enzymatic activities. Which amino acids
require a related enzyme complex, and what is the name of the
enzyme?
The branched-chain amino acids leucine, isoleucine, valine and threonine. The required enzyme is the branched-chain α-ketoacid dehydrogenase complex.
The carbon skeletons of the 20 common amino acids
can be degraded into a limited number of end products. What are the
end products and in what metabolic pathway are they commonly
found?
. Pyruvate (glycolysis and gluconeogenesis), acetyl CoA (citric acid cycle and fatty acid synthesis), acetoacetyl CoA (ketone-body formation), α-ketoglutarate (citric acid cycle), succinyl CoA (citric acid cycle), fumarate (citric acid cycle), and oxaloacetate (citric acid cycle and gluconeogenesis).
Pyruvate carboxylase deficiency is a fatal
disorder. Patients with pyruvate carboxylase deficiency sometimes
display some or all of the following symptoms: lactic acidosis,
hyperammonemia (excess NH4+ in the blood), hypoglycemia,
and demyelination of the regions of the brain due to insufficient lipid
synthesis. Provide a possible biochemical rationale for each of these
observations.
The precise cause of all of the symptoms is not firmly established, but a likely explanation depends on the centrality of oxaloacetate to metabolism. A lack of pyruvate carboxylase would reduce the amount of oxaloacetate. The lack of oxaloacetate would reduce the activity of the citric acid cycle and so ATP would be generated by lactic acid formation. If the concentration of oxaloacetate is low, aspartate cannot be formed and the urea cycle would be compromised. Oxaloacetate is also required to form citrate, which transports acetyl CoA to the cytoplasm for fatty acid synthesis. Finally, oxaloacetate is required for gluconeogenesis.
Pyridoxal phosphate is an important coenzyme in
transamination reactions. We have seen this coenzyme before, in
glycogen metabolism. Which enzyme in glycogen metabolism
requires pyridoxal phosphate and what role does the coenzyme play
in this enzyme?
Glycogen phosphorylase. The coenzyme serves as an acid base catalyst..
The glucose-alanine cycle is
reminiscent of the Cori cycle, but the glucose-alanine cycle can be
said to be more energy efficient. Explain why this is so.
In the Cori cycle, the carbon atoms are transferred from muscle to liver as lactate. For lactate to be of any use, it must be reduced to pyruvate. This reduction requires high-energy electrons in the form of NADH. When the carbon atoms are transferred as alanine, transamination yields pyruvate directly.
Another helping hand. In eukaryotes, the 20S proteasome component
in conjunction with the 19S component degrades ubiquitinated
proteins with the hydrolysis of a molecule of ATP. Archaea lack
ubiquitin and the 26S proteasome but do contain a 20S proteasome.
Some archaea also contain an ATPase that is homologous to the
ATPases of the eukaryotic 19S component. This archaeal ATPase
activity was isolated as a 650-kDa complex (called PAN) from the
archaeon Thermoplasma, and experiments were performed to
determine if PAN could enhance the activity of the 20S proteasome
from Thermoplasma as well as other 20S proteasomes.
Protein degradation was measured as a function of time and in the presence of various combinations of components. Graph A shows
the results.

a. What is the effect of PAN on archaeal proteasome activity in the
absence of nucleotides?
b. What is the nucleotide requirement for protein digestion?
c. What evidence suggests that ATP hydrolysis, and not just the
presence of ATP, is required for digestion?
A similar experiment was performed with a small peptide as a
substrate for the proteasome instead of a protein. The results
obtained are shown in graph B.
d. How do the requirements for peptide digestion differ from those of
protein digestion?
e. Suggest some reasons for the difference.
The ability of PAN from the archaeon Thermoplasma to support
protein degradation by the 20S proteasomes from the archaeon
Methanosarcina and rabbit muscle was then examined.
Percentage of digestion of protein substrate (Source of the 20S
proteasome)
Additions Thermoplasma Methanosarcina R
f. Can the Thermoplasma PAN augment protein digestion by the
proteasomes from other organisms?
g. What is the significance of the stimulation of rabbit muscle
proteasome by Thermoplasma PAN?
a. Virtually no digestion in the absence of nucleotides. b. Protein digestion is greatly stimulated by the presence of ATP. c. AMP-PNP, a nonhydrolyzable analog of ATP, is no more effective than ADP. d. The proteasome requires neither ATP nor PAN to digest small substrates. e. PAN and ATP hydrolysis may be required to unfold the peptide and translocate it into the proteasome. f. Although Thermoplasma PAN is not as effective with the other proteasomes, it nonetheless results in threefold to fourfold stimulation of digestion. g. In light of the fact that the archaea and eukarya diverged several billion years ago, the fact that Thermoplasma PAN can stimulate the rabbit muscle proteasome suggests homology not only between the proteasomes, but also between PAN and the 19S subunit (most likely the ATPases) of the mammalian 26S proteasome.
Identify the means by which protein turnover is regulated.
Protein turnover is determined by the rates of synthesis and degradation, and cells can therefore govern protein abundance by controlling these opposing cellular processes.
Describe the common features of amino acid degradation.
What is the degradation of amino acids?
Generally the first step in the breakdown of amino acids is the removal of the amino group, usually through a reaction known as transamination. The carbon skeletons of the amino acids undergo further reactions to form compounds that can either be used for the synthesis of glucose or the synthesis of ketone bodies.
Account for the fact that the symptoms of methylmalonic acidemia
can be so variable, from benign to fatal.
. Methylmalonic acidemia is an inherited disorder in which the body is unable to process certain proteins and fats (lipids) properly. The effects of methylmalonic acidemia, which usually appear in early infancy, vary from mild to life-threatening
Write out a complete mechanism for the
conversion of serine into aminoacrylate catalyzed by serine
dehydratase.
FOTİ VAR
The nervous system contains a substantial amount
of D-serine, which is generated from L-serine by serine racemase, a
PLP-dependent enzyme. Propose a mechanism for this reaction.
What is the equilibrium constant for the reaction L-serine⇌ Dserine?
The equilibrium constant for the interconversion of L-serine and Dserine is exactly 1.
In Chapter 8, we learned that there are two types
of bisubstrate reactions, sequential and double-displacement. Which
type characterizes the action of aminotransferases? Explain your
answer.
Double-displacement. A substituted enzyme intermediate is formed.
Degradation signals are commonly located in protein
regions that also facilitate protein-protein interactions. Explain why
this coexistence of two functions in the same domain might be
useful.
Exposure of such a domain suggests that a component of a multiprotein complex has failed to form properly or that one component has been synthesized in excess. This exposure leads to rapid degradation and the restoration of appropriate stoichiometries.
Within a few days after a fast begins, nitrogen excretion
accelerates to a higher-than-normal level. After a few weeks, the rate
of nitrogen excretion falls to a lower level and continues at this low
rate. However, after the fat stores have been depleted, nitrogen
excretion rises to a high level. ,
a. What events trigger the initial surge of nitrogen excretion?
b. Why does nitrogen excretion fall after several weeks of fasting?
c. Explain the increase in nitrogen excretion when the lipid stores
have been depleted.
a. Depletion of glycogen stores. When they are gone, proteins must be degraded to meet the glucose needs of the brain. The resulting amino acids are deaminated, and the nitrogen atoms are excreted as urea. b. The brain has adapted to the use of ketone bodies, which are derived from fatty acid catabolism. In other words, the brain is being powered by fatty acid breakdown. c. When the glycogen and lipid stores are gone, the only available energy source is protein.
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