Download Chapter 18 Homework Assignment Chapter 18 Amino Acid

Cellular Respiration
Chapter 18
Homework Assignment
Overview
• Stage 1: Acetyl-CoA Production
from:
• The following problems will be due once we finish
the chapter:
– Glucose (CHP 14)
– Fatty Acids (CHP 17)
– Amino Acids
1 6,
1,
6 10,
10 11,
11 13
13, 14
• Stage 2: Acetyl-CoA Oxidation
(TCA cycle; CHP 16)
• Stage 3: Electron Transfer &
Oxidative Phosphorylation (CHP 19)
• Additional Problems:
– Write out the five reaction steps of the urea cycle, using structures
to describe the intermediates. Use the correct stoichiometry to
show the final products derived from one ammonia molecule.
Identify the enzyme and any required cofactors for each step. Use
arrows to show which reactions are irreversible and which are
reversible
Chapter 18
1
Chapter 18
Amino Acid Oxidation &
the Production of Urea
Chapter 18
3
Amino Acid Metabolism
• Sources and destinations of amino acids
– Dietary and endogenous turnover
– Non-essential amino acids – biosynthesis
• Extracting the energy – carbon catabolism
– Carbon entry into the TCA cycle
– One-carbon
One carbon transfer reactions
– Glucogenic and ketogenic amino acids
• Managing the ammonia – nitrogen metabolism
– From tissues to liver
– Reactions in the liver – the urea cycle
– Integrative pathways – TCA, urea, and gluconeogenesis
Chapter 18
4
1
Amino Acid Metabolism
Amino Acid Metabolism
Where Do Amino Acids Come From?
Where Do They Go?
Alternative Fates of Amino Acids
• Sources of amino acids
– Dietary ~100 gm/day (“good” vs. “bad” protein)
– Endogenous protein turnover 300-600 gm/day
• Use it or lose it – amino acids can’t be stored
–
–
–
–
Protein biosynthesis
Conversion to essential metabolites
Oxidation for energy
Excretion
Chapter 18
5
Amino Acid Metabolism
7
The Ten Amino Acids We Can Make
•
The hormone gastrin, secreted in response to
stomach protein, stimulates HCl release
(lowering stomach pH) and pepsinogen
secretion, denaturing proteins and activating
pepsin from its zymogen
•
The hormone secretin (stimulated by low pH)
causes the pancreas to release bicarbonate, to
bring pH up to 7
•
The hormone cholecystokinin (responding to
amino acids) stimulates pancreatic release of
protease zymogens, including trypsinogen and
chymotrypsinogen
•
These and other proteases are activated and
digest most proteins to free amino acids (some
fibrous proteins like keratin are resistant)
On to the liver…
Chapter 18
Amino Acid Metabolism
Protein in the Diet
Chapter 18
• Many amino acids are absorbed
and used directly - as is - in tissue
protein synthesis.
• For humans, 10 of the 20 natural
amino acids are “essential”,, and
must be obtained from the diet
• Excess amino acids cannot be
stored, but can be oxidized for
energy – carnivores derive up to
90% of their energy needs from
amino acid oxidation (for people
it’s 10-15%)
6
• The rest,, we
must ingest…
Cys, Gly, Ser come from
3-phosphoglycerate
Chapter 18
8
2
Amino Acid Metabolism
Amino Acid Metabolism
Nitrogen and Carbon Go Separate Ways
Sources of Stored Metabolic Energy
For a 70 kg male after overnight fasting…
% of cal
mass
• Glycogen – liver:
80 gm
0.2%
• Glycogen – muscle:
150 gm
0.4%
• Fat:
15,000 gm
85%
• Protein:
6,000 gm
14.5%
But, is the protein really “stored”?
Chapter 18
9
11
Amino Acid Metabolism
Amino Acid Metabolism
Amino Acids Not Used in Biosynthetic
Reactions Undergo Oxidative Degradation
• Amino acids as fuel may be derived from
– A diet rich in protein
– Cellular protein turnover
– Abnormal
Ab
l protein
t i turnover
t
(starvation
( t
ti or diabetes)
di b t )
• Energy comes from the α-keto acid carbon
skeleton, after removal of the amino group, into
either the TCA cycle or gluconeogenesis
• Removal of the amino group (waste) requires
expenditure of energy
Chapter 18
Fig. 18-1
Chapter 18
10
Nitrogen and Carbon Go Separate Ways
• The first step in the catabolism of most
Aas once in the liver is the removal of
the α-amino group
• This reaction is catalyzes by a group of
enzymes called the aminotransferases
or the transanimases
• In general, the amino group is
transferred from the AA to α-Keto –
glutarate, leaving behind the α-Keto
acid analog of the AA and producing Lglutamate
• So great. We started with an AA and
an α-Keto acid and we ended with an
AA and an α-Keto acid
Chapter 18
Futile Cycle?
12
3
Amino Acid Metabolism
Amino Acid Metabolism
Amino Acids and the TCA Cycle
Aminotransferase and PLP
• All aminotransferases have the same
prosthetic group and the same reaction
mechanism.
• They vary in their substrate specificity.
prosthetic g
group
p is py
pyridoxal
• The p
phosphate (PLP) that is derived from
vitamin B6
• PLP functions as an intermediate carrier
of the amino group at the active site
• This group is usually covalently attached
to the enzyme via a linkage to the εamino group of a Lys residue
• Note a curious
fact: each
entry point for
the catabolism
of amino acid
backbones
follows an
oxidation step
in the cycle
Chapter 18
13
Amino Acid Metabolism
Fate of Amino Acid Carbon Skeletons
• The carbons of the 20 amino acids converge into the TCA
cycle (multiple entries give 28 paths)
• In fact, they enter it at only 5 places after each oxidation
step in the cycle
• Carbons from larger amino acids may enter the TCA cycle
at more than one place
5
4
4
Chapter 18
– Phe, Tyr, Leu, and Ile at 2 places (note relationships!)
– Trp (and Thr) can enter at 3 places
2
6
15
Amino Acid Metabolism
28 Precursors for Carbon Entry to TCA
5
Chapter 18
• The cell employs 2 reactions frequently:
– Transaminations
2
– One-carbon transfers
14
• All the reactions occur in the liver, except for the branchedchain amino acids: leucine, isoleucine, and valine
Chapter 18
16
4
Amino Acid Metabolism
Amino Acid Metabolism
Glucogenic and Ketogenic Amino Acids
Where Carbon Skeletons from Amino
Acids Can Enter the TCA Cycle
Asn & Asp
• Carbons from the pink
amino acids can all
participate in gluconeogenesis
• Carbons from the blue
amino acids can all end
up as acetoacetyl-CoA
and or acetyl-CoA
Oxaloacetate
Tyr, Phe,Trp, Leu & Lys
Acetoacetyl~CoA
Met, Val, Ile, Thr
Succinyl~CoA
Gln, His, Arg, Pro & Glu
α-Ketoglutarate
Ala, Ser, Cys, Gly, Thr & Tyr Pyruvate
Ile, Leu, Thr & Trp
Acetyl-CoA
Tyr & Phe
Fumarate
– If in excess, these can
produce ketone bodies
You don’t have to memorize these, but you should know
the identity of the 5 main entry points to the TCA.
Chapter 18
17
Amino Acid Metabolism
19
Amino Acid Metabolism
Extrahepatic Metabolism of Val, Ile, and Leu
• The branched-chain α-keto
acid dehydrogenase
complex catalyzes oxidative
decarboxylation (Hmm….
That sounds familiar!
– How many cofactors? How
many enzymes?
?
• The enzyme is
phosphorylated (inactive)
unless excess branched AA’s
are present;
• If defective, the α-keto acids
accumulate, and spill over
into the urine
Chapter 18
Chapter 18
• Trp, Phe, Tyr, Ile are in
both categories
• Only Leu and Lys are
solely ketogenic
18
Can You Now Answer These Questions?
• Why are there only 20 amino acids in proteins, but
28 “paths” into the TCA cycle?
• For many of these “paths” the stoichiometry of
carbon atoms does not add up: can you name 3
different ways that these carbons are directed
elsewhere?
• What is a “glucogenic” amino acid?
• What is a “ketogenic” amino acid?
• What other pathway interfaces with the latter?
• Can an amino acid be both glucogenic and
ketogenic? Use an example to explain.
Chapter 18
20
5
Amino Acid Metabolism
Amino Acid Metabolism
Summary - Fate of the Carbons
Nitrogen Removal in the Liver
• Carbon skeletons end up in TCA where they can be
ketogenic or glucogenic (or both)
• Leu, Ile, and Val are degraded extrahepatically
• Amino acids only undergo partial oxidation in the liver, but
this can fulfill the “fed” liver ATP needs for both making
urea and
d ffor gluconeogenesis
l
i
• Thus, gluconeogenesis and urea synthesis can be
considered parts of the same pathway, integrated with the
TCA cycle: high urea synthesis reflects a high need for
glucose
• As with amino acid carbon catabolism, genetic defects in
the urea cycle can have serious consequences.
Chapter 18
21
•
After transfer of the amino group to αketoglutarate, the produced Lglutamate carries the group to the liver.
•
In the liver, GLU is transported into the
MT where it undergoes oxidative
deamination
•
This reaction is catalyzed by
glutamate dehydrogenase (MM 330
kDa) and produces NADH OR NADPH,
depending on which electron carrier is
used by the enzyme
•
The α-ketoglutarate can then be used
in the TCA cycle
•
The NH4+ has another fate….
Chapter 18
23
Amino Acid Metabolism
Amino Acid Metabolism
Where Does Nitrogen Go & How Does It Get There?
Aspartate Aminotransferase (AST)
• PLP (red) is in the
active site of one of
the two subunits of
this dimeric transaminase
• There are many
isozymes that are
each named after the
amino group donor
Transamination
PDB: 1AJS
Chapter 18
22
Chapter 18
24
6
Amino Acid Metabolism
Amino Acid Metabolism
Another Way to Get Ammonia to the Liver:
Glucose-Alanine Cycle
Glutamate & Glutamine are Important
Nitrogen Carriers
• Most amino acid metabolism occurs in the liver
• In the hepatocyte cytosol, α-ketoglutarate
receives amino groups transferred from other
amino acids,
acids to form glutamate (transamination)
• Glutamine is the common ammonia carrier from
other tissues by transamination of glutamate
(except muscle, where alanine is used – why?)
• Glutamate and glutamine then pass into liver
mitochondria for further metabolism
Chapter 18
25
Amino Acid Metabolism
Anerobic muscle contraction yields pyruvate
(from glycolysis) and amino groups (from
protein breakdown)
•
Alanine aminotransferase takes these two
products and makes alanine
•
The Al
Th
Ala th
then goes tto the
th liver
li
where
h
it iis
reconverted to pyruvate (and then to glucose,
via gluconeogenesis, to be exported back to
muscle)
•
This complements the Cori cycle with lactate
(see Box 15-1)
•
Chapter 18
The leftover ammonia is excreted from the liver
Why does this make metabolic sense
for liver and muscle function? 27
Amino Acid Metabolism
So, what about the ammonia?
Transport by Glutamine in the Bloodstream
• The enzyme glutamine synthetase,
which plays a central metabolic role in all
organisms, can transfer free ammonia to
glutamate, thus producing glutamine
• This two-step reaction (another one!)
requires an activated phosphorylated
intermediate
• Glutamine thus carries two amino
groups, is nontoxic and highly soluble,
and is present in blood at higher levels
than other amino acids
• Once in mitochondria, glutaminase
regenerates glutamate and ammonia
Chapter 18
•
26
• High levels of NH4+ are toxic to the central nervous
system - producing coma and death.
• Therefore, its levels are highly regulated
• What processes do you think would produce ammonia?
g rid of excess NH4+ from the brain may
y reduce α• Getting
ketoglutarate levels and inhibit the TCA cycle.
• Moreover, glutamine and its cousin γ-amino-butyric acid
(GABA) are neurotransmitters.
• The brain needs to maintain [ATP] at very high levels.
Why?
Chapter 18
28
7
Amino Acid Metabolism
Amino Acid Metabolism
Amino Acids vs. Urea vs. Ammonia
• “Normal” plasma levels in N-equivalents
How to Get Rid of Toxic Ammonia?
Amino acids
Glutamine
Alanine
(ingested protein)
(muscle and
other tissues)
(muscle)
Only urea
output is
proportional
to protein in Fig. 18-2b
the diet
Ammonium ion
Mmol-N/day
(gm/day)
40
(0.7)
Urea
800
(25)
Uric acid
micromolar
L-Glutamic Acid
L-Glutamine
L-Alanine
Urea
NH3
Creatinine
15
(0.8)
These values demonstrate the efficiency of the
conversion of ammonia to urea. Luckily for us!
40
(1.5)
Chapter 18
29
Chapter 18
31
Once Safely in the
Liver, how is
Ammonia Converted
to Urea for Excretion?
Amino Acid Metabolism
What’s So Great About Urea?
• Urea synthesis is essential to humans, because
– We require protein in our diet to provide amino acids
– These AAs can’t be stored, unlike fats and carbohydrates
•
– The resultant excess nitrogen after conversion to ammonia must be
excreted as urea
Urea synthesis occurs nearly
exclusively in the liver
•
The ammonia deposited in the
MT of hepatocytes is converted
t urea in
to
i the
th Urea
U
C
Cycle
l
•
The urea is produced then
passes into the bloodstream
and on to the kidneys where it is
excreted into the ……?
•
So, the liver will take care of the
ammonia for us, but its going to
cost us…. (doesn’t everything?)
– If not,
not ut will circulate in the bloodstream and be toxic if it rises above
certain levels
• Urea is compact (and symmetric),
– 2 of its 8 atoms are nitrogen (what % by weight?)
– It is very water soluble while being nontoxic
• Urea is not so great for birds and terrestrial reptiles
– Excretion requires large water loss, so these “uricoteles” eliminate
crystalline uric acid
Chapter 18
103
554
436
9,200
22 (10-50)
30
Chapter 18
32
8
Amino Acid Metabolism
Step 1: Formation of Carbamoyl Phosphate
• In the mitochondrial matrix, the
enzyme carbamoyl phosphate
synthetase I condenses bicarbonate,
ammonia, and a phosphoryl group
• This results in the formation of
carbamoyl phosphate
• This compound can enter the urea
cycle by condensing with ornithine (an
amino acid!)
Amino Acid Metabolism
Step 2 : Citrulline to Argininosuccinate
• Ornithine transcarbamoylase
(OTCase), in the MT matrix,
condenses CP with ornithine to form
citrulline (also an AA)
• Citrulline transits to cytosol
• In the cytosol,
cytosol Citrulline is activated by
ATP, forming an AMP-Citrulline
intermediate
– This reaction (and the next!) is catalyzed
by argininosuccinate synthetase
• The intermediate is then condensed
with aspartate (the source of the
second amino group)
Chapter 18
33
Amino Acid Metabolism
Chapter 18
Amino Acid Metabolism
Steps 2 – 4: The Urea Cycle
Steps 3 & 4: Urea Production
• The UC scavenges
nitrogen from both MT
matrix and the cytosol
(from aspartate)
• The 5 enzymes
involved are clustered,
and employ substrate
channeling
• Only urea is released
into the cytosolic pool
of metabolites
Chapter 18
35
• Argininosuccinate is cleaved by
argininosuccinate lyase to yield free
arginine and fumarate
• Arginine is then cleaved by arginase
to yield urea and ornithine
• Urea is carried to the kidneys and
excreted
• Ornithine then re-enters mitochondria
via ORNT1 antiporter, in exchange for
citrulline export
34
Chapter 18
36
9
Amino Acid Metabolism
Amino Acid
Metabolism
Regulation of Glutamine Synthetase
So, how much is that
going to cost me?
• The importance of this
enzyme is reflected in
the complexity of its
regulation
g
((the E. coli
enzyme is shown).
• Conversion of ammonia to
Carbamoyl phosphate
costs 2 ATP
• Conversion of Citrulline to
Argininosuccinate costs 1
ATP
• So we are talking 3 ATPs
to remove one ammonia,
but it is worth it!
Chapter 18
And that’s all you need
to know…
37
Amino Acid Metabolism
Chapter 18
Amino Acid Metabolism
Regulation of the Urea Cycle
Can You Now Answer These Questions?
What two situations might result
in an increase in urea production?
• What transports nitrogen from amino groups to the liver?
• Why must ammonia be transported to the liver (and kidney)
and how is it done?
• How do the Cori cycle and the glucose-alanine cycle work
together?
• What happens to excess nitrogen once in the liver?
• How does the kidney counteract acidosis (remember ketone
bodies…)?
• Do you know the α-ketoacid counterparts for alanine,
glutamate, and aspartate?
• What two reasons may account for the special toxicity of
ammonia in the brain?
Chapter 18
39
38
• Increased [glutamate] in the MT
matrix signals excess nitrogen
which results in both genetic and
allosteric regulation
Arg • On an allosteric level, this situation
will:
(except in
mammals)
Chapter 18
– Drive the synthesis of Nacetylglutamate which acts as an up
regulator of CPS-I
• On a genetic level, this situation
will up-regulate the production
levels of CPS-I and the 4 urea
40
cycle enzymes
10
Amino Acid Metabolism
Amino Acid Metabolism
The Urea Cycle Intersects the TCA Cycle
•
Because fumarate produced in the
argininosuccinase reaction is also an
intermediate in the TCA cycle, the two cycles
have been dubbed the “Krebs Bicycle”
•
However, the two cycles operate
independently and communication between
them depends on the transport of key
intermediates between the MT and the
cytosol (WHY??)
There are isozymes in the cytosol that allow for connection to the TCA cycle via
the Aspartate- Argininosuccinate shunt
•
•
Current thinking:
–
Excess matrix nitrogen (as glutamate) can be “dumped” as aspartate (by transamination – of
what?) to the cytosol into the urea cycle
–
Cytosolic fumarate itself cannot re-enter the matrix, making the “bicycle” impossible until fumarate
is made into malate
– The ORNT1 antiport transporter brings ornithine into the matrix, exchanging citrulline to the cytosol
Chapter 18
41
Amino Acid Metabolism
Sir Archibald Garrod discoved alkaptonuria as a genetic disease, which was
the beginning of our understanding of
“inborn errors of metabolism”…
• Alkaptonuria
– A defect in amino acid catabolism causes
your urine to turn black
– Defective homogentisate 1,2-dioxygenase
• Tyrosinemia I
– Defective fumaryl-acetoacetase
Chapter 18
43
Amino Acid Metabolism
Summary – Fate of the Nitrogen
• Amino group is removed by transamination to αketoglutarate or OAA
• Glutamate dehydrogenase gives NH4+ (mito)
• NH4+ is converted to urea in its eponymous cycle
• Which is restricted to the liver (other tissues lack
arginase)
• Urea cycle is interconnected with the TCA cycle
• Urea cycle enzymes are regulated at the level of enzyme
synthesis.
• Flux through the urea cycle at the level of carbamoyl
phosphate synthesis can be regulated allosterically by Nacetylglutamate.
Chapter 18
Diseases of AA Carbon
Catabolism
42
Diseases of AA CarbonFig. 18-23
Catabolism
• Phenylketonuria:
– Defective phenyl-alanine
hydroxylase
– Accumulation of Phe and
phenylpyruvate and
phenyllactate is deleterious
– Where might these last two
come from? (Hint: see Fig.
18-25)
Chapter 18
44
11
Amino Acid Metabolism
Other Diseases of Amino Acid
Carbon Catabolism
• Albinism
– Defective tyrosine 3-mono-oxygenase
– Melanin synthesis from tyrosine is blocked
– Affected individuals lack skin pigmentation
• Maple syrup urine disease
– Branched-chain α-ketoacid dehydrogenase (Ile, Leu,
Val)
• Methylmalonic acidemia
– Methylmalonyl-CoA mutase (conversion of propionylCoA to succinyl-CoA)
Chapter 18
45
12