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Biochemistry
Protein Turnover and Amino Acid
Catabolism
Stryer Ch. 23
Protein Turnover
•
Proteins are constantly being degraded and
resynthesized
– Location of protein degradation
• Proteins from diet are hydrolyzed in the
digestive tract
• Proteins within each cell are broken down
within that cell in a proteasome
•
EXCESS AMINO ACIDS ARE NEITHER
STORED NOR EXCRETED
– Degradation of amino acids requires
• Removal of amino group – deamination
–
Urea cycle
• Conversion of carbon skeletons into glucose /
glycogen via conversion to:
–
–
–
–
•
Acetyl CoA
Acetoacetyl CoA
Pyruvate
Citric Cycle intermediate
Amino Acids are classified as essential and
nonessential
– Essential – organism lacks the ability to
synthesize these amino acids; must be acquire
from diet
– Nonessential – amino acids are synthesized in
vivo from smaller non amino acid precursors
Degradation of Dietary Proteins
• Dietary Proteins are digested by general and specific proteases
into free amino acids, dipeptides and tripeptides, which are
absorbed into the intestine by specific transporters.
– Stomach
• Acidic pH = 2
• Pepsin – primary protease of stomach
– Lumen of Intestine
• MOST enzymes involved in digesting proteins are synthesized in the pancreas
and secreted as zymogens.
• Aminopeptidase – nonspecific protease that sequentially hydrolyze proteins
from the amino terminal end.
Degradation of Cellular Proteins
• Proteins turn over within the cell.
– Damaged / Mis-folded proteins are quickly destroyed
• Identified by attachment of ubiquitin
– Proteins that are no longer needed
• Half life of protein varies from protein to protein.
– Minutes, hours, days, years, decades.
– Determined by N-terminal residue
• R or L favors ubiquination (fast destruction)
• M or P disfavors ubiquination (slow destruction; long half life)
– Other signals exist.
• Some diseases are due to abnormal aggregation /
improper destruction of proteins.
Ubiquitin (Ub)
• 8.5 kd protein
– 76 amino acids
• Found in ALL eukaryotes
• “Mark of death”
– tags protein for proteolysis
• Ub covalently attached to proteins
– C-terminal gly of Ub
– Lys of protein
• ε amino group (R-group)
• Ub forms polymers
– polyubiquitination
Ub attachment requires three enzymes
• E1 or Ubiquitin-activating enzyme
– Activates Ub by attachment to AMP
– Links C-terminal carboxylate of Ub to sulfhydryl of E1 by thioester.
• E2 or Ubiquitin-Conjugating enzyme
– activated Ub transferred from sulfhydryl of E1 to sulfhydryl of E2
• E3 or ubiquitin-protein ligase
– Catalyzes the transfer of Ub from E2 to target protein
– Largest gene family in humans
Disease linked to E3
• Parkinson disease (some forms)
– Improper functioning E3 leads to abnormal
accumulation of proteins.
• Angelman syndrome
– Severe neurological disorder (mental retardation,
absence of speech, uncoordinated movement.)
• Human papilloma virus (HPV)
– Causes >90% of cervical carcinomas
– Activates specific E3 of host leading to destruction of
p53 (tumor suppressor) and other DNA repair genes.
Structure of Proteasome
• 26 S Large protease complex
– Two 19S caps
• Contains AAA class ATPase activity
– ATPase associated with various
activities
– One 20S catalytic core
• Four rings of 7 subunits each
– 28 subunits total
» 14 alpha (outer rings)
Seven isoforms of alpha are known
» 14 beta (two inner rings)
Seven isoforms of beta are known.
• Active sites are on beta subunits.
Action of Proteasome
• Hydrolyzes Ubiquinated proteins.
• Proteolytic active sites are
located on the beta subunits
– Threonine residue acts as
nucleophile to attach carbonyl of
peptide bond.
• Digest proteins to 7 – 9 amino
acid peptides
– These peptides are released from
proteasome and further degraded to
amino acids by cellular proteases.
Bortizomib (Velcade)
• Inhibitor of proteasome
• Therapy for multiple
myeloma
Nitrogen Removal from Amino Acids
• Liver is primary site of amino acid degradation
– Muscle is secondary site of amino acid degradation for
branched chain amino acids (e.g. L,I,V)
• Two step process
Removal of Amino Groups
•
Aminotransferase (AKA transaminases)
– α amino group transferred to αketoglutarate to form glutamate
– Examples
• Aspartate amino transferase
• Alanine amino transferase
– Reversible
• Also used in synthesis of amino acids.
– Contain pyridoxal phosphate (PLP) as
prosthetic group
• Derived from pyridoxine (vitamin B6)
•
Glutamate dehydrogenase
– Converts nitrogen of glutamate to
ketoacid and free ammonium ion
– Localized to mt of Liver
– May use EITHER NAD+ or NADP+
– Close to equilibrium in liver
• Direction of reaction determined by
[substrate] or [product]
• Normally driven forward by removal
of ammonium
Pyridoxal Phosphate (PLP)
• Prosthetic group
• Group transfer to or from
an amino acid.
• Precursor = Pyridoxine
(Vitamin B6)
• Deficiency = depression,
confusion, convulsions.
Pyridoxal Phosphate (Cont’)
• Mechanism see p 659
– 660.
Serine and Threonine are directly deaminated
• Serine dehydratase
– dehydrates and deaminates serine to
produce pyruvate and ammonium
• Amino group is NOT transferred to αketoglutarate
• Threonine dehydratase
– dehydrates then deaminates threonine
to yield α-ketobutyrate and
ammonium
• Amino group is NOT transferred to αketoglutarate
Peripheral Tissues Transport Nitrogen to the
Liver Glucose – Alanine Cycle
• Muscle tissue uses
branched amino acids
as fuel.
• The nitrogen from
these amino acids is
transferred to alanine
(through glutamate).
• Alanine is carried to
liver via blood stream.
• Alanine is converted
to pyruvate which is
used to produce
glucose.
Excretion of Nitrogen
• Terrestrial vertebrates
– Ureotelic – excrete excess nitrogen as urea
• Aquatic vertebrates and invertibrates
– Ammoniotelic – excrete excess nitrogen as ammonium
• Birds and reptiles
– Uricotelic – excrete excess nitrogen as uric acid
Urea Cycle
Hans Krebs and Kurt Henseleit (1932)
• Cycle responsible for synthesis of
urea
– Urea = form of nitrogen excreted in
vertebrates.
– Humans excrete 10 kg urea / yr.
• 10 kg = 22 lbs.
• Source of atoms in Urea
– 1 N from free NH4+
• ammonium
– C from HCO3• bicarbonate derived from hydration
of CO2
– 1 N from aspartate
• Carbamoyl Phosphate
– intermediate in urea cycle
– Synthesized from NH3 and HCO3– Carbamoyl group has a high
phosphate transfer potential due to
anhydride bond.
Carbamoyl Phosphate Synthetase
• Catalyzes the three step synthesis of carbamoyl phosphate
– Bicarbonate phosphoryled by phosphate from ATP forming
carboxyphosphate.
– Carboxyphosphate reacts with ammonia to form carbamic acid.
– Carbamic acid is phosphorylated by ATP to yield carbamoyl phosphate.
• Mt matrix
• 2 ATP / carbamoyl phosphate synthesized
• Isozyme catalyzes the synthesis of carbamoyl phosphate for use in
pyrimidine biosynthesis
Carbamoyl Phosphate Synthetase
• Three reaction sites
– Glutamine hydrolysis site
– Bicarbonate phosphorylation site
– Carbamic acid phosphorylation site
• Substrate channeling
– Substrates pass from one active site
to another active site through a
channel. Is not released by enzyme.
– Benefits
• Increases the rate of reaction because the
substrate is not “released” from the
enzyme.
• Protects libile substrates from
degradation by hydrolysis
– Carbonic acid decomposes in 1 s at ph
=7.
Ornithine transcarbamoylase
• Catalyzes the synthesis of citrulline from ornithine and carbamoyl
phosphate
• Citrulline and ornithine
– Amino acids NOT used in synthesis of proteins.
• Mt matrix
• Following synthesis, citrulline is transported to cytoplasm
Argininosuccinate Synthetase
• Catalyzes the condensation of citrulline with aspartate to
form argininosuccinate
• Cytoplasm
• 1 ATP / argininosuccinate synthesized
– Hydrolized to AMP and PPi
Argininosuccinase
• Cleaves argininosuccinate into arginine and fumarate.
– Conserves carbon skeleton of aspartate in fumarate.
• Cytoplasm
Arginase
• Hydrolyzes arginine into ornithine and urea.
• Ornithine transported into mt
• Urea excreted.
Urea Cycle is Linked to Gluconeogenesis
• Aspartate and Fumarate link Urea Cycle to
Gluconeogenesis.
Inherited Diseases of the Urea Cycle
• Hyperammonemia
– Elevated levels of ammonium (NH4+) in blood
– Causes include:
• lack / reduced synthesis of carbamoyl phosphate
• Lack / reduced activity of the four enzymes of urea synthesis
• Why is excess ammonium toxic?
– Maybe the synthesis / accumulation of glutamine causes an
osmotic imbalance???
Treatments of Hyperammonemias
• Argininosuccinase deficiency
treatment
– Provide excess arginine in diet
– Restrict total protein in diet
• reduces amount of nitrogen to be excreted
• Arginine is converted into
Ornithine which reacts with
Carbamyol phosphate to form
citrulline. Citrulline reacts with
aspartate to form arginiosuccinate
which is excreted.
Treatments of Hyperammonemias (cont’)
• Carbamoyl phosphate synthetase or ornithine transcarbamoylase deficiency
treatment
– Excess nitrogen accumulates in glycine and glutamine.
– Restrict protein in diet
– Supplement diet with benzoate and phenylacetate
• Converted into Benzoyl CoA and phenylacetate CoA
– Excess nitrogen excreted as hippurate or phenylacetylglutamine
Fates of Carbon Skeletons
• Carbon skeletons from deaminated amino acids are converted into
seven intermediates
–
–
–
–
–
–
–
Pyruvate
Acetyl CoA - K
Acetoacetyl CoA - K
α-Ketoglutarate
Succinyl CoA
Fumarate
Oxaloacetate
• Ketogenic Amino Acids
– Carbon skeletons are converted into intermediates (acetyl CoA or
Acetoacetyl CoA) that can form ketone bodies or fatty acids
• Glucogenic Amino Acids
– Carbon skeletons are converted into intermediates that can be used to
synthesize glucose.
• Both
– Some amino acids have carbons that end up in ketogenic and glucogenic
intermediates.
Fates of Carbon Skeletons
Amino Acids
• GLUCOGENIC
BOTH
NONESSENTIAL
– Alanine (A, Ala)
– Arginine (R, Arg)*
– Asparagine (N,Asn )
– Aspartate (D, Asp)
– Cysteine (C, Cys)
– Glutamate (E, Glu)
– Glutamine (Q, Gln)
– Glycine (G, Gly)
– Proline (P, Pro)
– Serine (S, Ser)
NONESSENTIAL
– Tyrosine (Y, Tyr)
ESSENTIAL
ESSENTIAL
(Val and His Three Methods)
(Iley Trpped BOTH Phesants)
–
–
–
–
– Isoleucine (I, ile)
– Phenylalanine (F, Phe)
– Tryptophane (W, Trp)
Histidine (H, His)*
Methionine (M, Met)
Threonin (T, Thr)
Valine (V, Val)
Essential: TV FILM HW(R)K
KETOGENIC
NONESSENTAIL
ESSENTAIL
KETONES in Leu of Lysine
--Leucine (L, Leu)
-- Lysine (K, Lys)
Updated 2014
Amino Acids
• GLUCOGENIC
BOTH
NONESSENTIAL
– A
– R*
– N
– D
– C
– E
– Q
– G
– P
– S
NONESSENTIAL
– Y
ESSENTIAL
ESSENTIAL
(Val and His Three Methods)
(Iley Trpped BOTH Phesants)
–
–
–
–
– I, Ile
– F, Phe
– W, Trp
H, His*
M, Met
T, Thr
V, Val
Essential: TV FILM HW(R)K
KETOGENIC
NONESSENTAIL
ESSENTAIL
KETONES in Leu of Lysine
--L, Leu
--K, Lys
Updated 2014
Definitions
• Glucogenic Amino Acids
– Carbon skeletons are converted into intermediates that can be
used to synthesize glucose.
• Ketogenic Amino Acids
– Carbon skeletons are converted into intermediates (acetyl CoA
or Acetoacetyl CoA) that can form ketone bodies or fatty acids
– NOT substrates for glyconeogenesis
• Nonessential Amino Acids
– enzymes present for de novo synthesis of these amino acids
• Essential Amino Acids
– lacks enzymes to synthesize the amino acids.
– Must be obtained from diet.
Pyruvate
• Amino Acids with 3 C
backbone
– Ala, Trp
– Ser, Gly
– Cys
• Sulfur is converted to
H2S, SCN- or SO32-
– Thr
• 2-amino-3-ketobutyrate
intermediate
Acetyl CoA
Acetoacetyl CoA
• Branched chain (Leu,
Ile, Lys,) and aromatic
amino acids (Phe, Trp,
Tyr)
Oxygenases are Required for
Degradation of Aromatic Amino acids
• Phenylalanine
hydroxylase
– Hydroxylates Phe to
Tyr
– Monooxygenase
• Each atom of oxygen
incorporated into
different products
– Tyr and water
– Tetrahydrobiopterin
• Electron carrier
Tetrahydrobiopterin (BH4)
•
Electron carrier
– Derived from Biopterin
•
Synthesized in vivo from phenylalanine
– NOT a vitamin because it is synthesized by the body.
•
Phenylalanine hydroxylase
– Synthesizes dihydrobiopterin by hydroxylating phenylalanine
•
Dihydrofolate reductase
– Reduces dihydrobiopterin to tetrahydrobiopterin using NADPH
•
Dihydropteridine reductase
– Reduces quinonoid dihydrobiopterin itto Tetrahydrobiopterin using NADPH
Phe / Tyr
• 1.Transamination
• 2. p-hydroxyphenylpyruvate hydroxylase
– Dioxygenase
• both atom of oxygen are incorporated into the product
• 3. Homogentisate oxidase
– Dioxygenase
• 4.Isomerization
• 5. Hydrolyzed
2
1
5
4
3
Trp
Converted into alanine and acetoacetate via the action of
several oxidases.
2
1
4
3
α-Ketoglutarate
• Amino acids with C5
skeletons
– Glu
• Deaminated into αKetoglutarate
– Gln
• Hydrolyzed to glutamate
by Glutaminase
– Pro
• Converted to glutamate γsemialdehyde
– Arg
• Converted to glutamate γsemialdehyde
– His
• Multistep process with 4imidazolone 5-propionate
intermediate
Succinyl CoA
• Major intermediates are common to oxidation of odd numbered fatty acids.
– Propionyl CoA
– Methylmalonyl CoA
– Succinyl CoA
• Met
– Nine steps involving S-adenosylmethionine (SAM) as methyl donor.
– See next slide
• Val
• Iso
Met degradation
Fumarate
• Asp
• Tyr
– Some carbons
• Phe
– Some carbons
Oxaloacetate
• Asp
– Direct deamination into Oxa
• Asn
– NH4+ removed by Asparaginase to
form Asp which is deaminated
Alcaptonuria
• Archibald Garrod (1902)
– First description of an inborn
error of metabolism
• Defective degradation of phe
and tyr.
• Recessive inheritance
• Homogentisate accumulates
and is excreted in the urine.
• Harmless condition
Maple Syrup Urine Disease
(branched-chain ketoaciduria)
• Defective / missing branchedchain dehydrogenase
• Defective degradation of val,
ile, leu.
– Causes elevation of the level of
these amino acids and the α
ketoacid derivatives in vivo and in
urine.
• Causes Mental and Physical
retardation
• Patients urine smells like maple
syrup.
• Detection
– Reaction of α ketoacid in urine
with 2,4-dinitrophenylhydrazine
– Mass Spec.
Phenylketonuria (PKU)
• Defective / missing phenylalanine
hydroxylase (or the tetrahydrobiopterin
cofactor)
– Phenylalanine cannot be converted into tyrosine
therefore phe accumulates
• Autosomal recessive
• Due to high [phe], reactions of Phe not found
in normal individuals are prevalent in PKU
patients
– e.g. formation of phenylpyruvate
• Phenylpyruvate in urine with FeCl3 to turn urine
olive green. (this reaction lead to the initial
description of PKU)
•
Untreated PKU leads to retardation and
reduced like expectancy (death in 20 – 30s).
• Treatment
– Low phenylalanine diet.
• Just enough phe for growth and replacement.
• Prevents accumulation of phe
• Must be started soon after birth.
Other Diseases involving Enzymes
of Amino Acid Degradation