1. Protein Turnover and Amino Acid Catabolism Stryer Ch. 23
Biochemistry
Protein Turnover and Amino Acid Catabolism
Stryer Ch. 23

2. 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

3. 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.

4. 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.

5. Ubiquitin (Ub) 8.5 kd protein Found in ALL eukaryotes “Mark of death”
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

6. 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

7. Disease linked to E3 Parkinson disease (some forms) Angelman syndrome
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.

8. 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.

9. 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.

10. Bortizomib (Velcade) Inhibitor of proteasome
Therapy for multiple myeloma

11. 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

12. Removal of Amino Groups
Aminotransferase (AKA transaminases)
α amino group transferred to α-ketoglutarate 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

13. Pyridoxal Phosphate (PLP)
Prosthetic group
Group transfer to or from an amino acid.
Precursor = Pyridoxine (Vitamin B6)
Deficiency = depression, confusion, convulsions.

14. Pyridoxal Phosphate (Cont’)
Mechanism see p 659 – 660.

15. Serine and Threonine are directly deaminated
Serine dehydratase
dehydrates and deaminates serine to produce pyruvate and ammonium
Amino group is NOT transferred to α-ketoglutarate
Threonine dehydratase
dehydrates then deaminates threonine to yield α-ketobutyrate and ammonium

16. 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.

17. 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

18. 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.

19. 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

20. 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.

21. 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

22. Argininosuccinate Synthetase
Catalyzes the condensation of citrulline with aspartate to form argininosuccinate
Cytoplasm
1 ATP / argininosuccinate synthesized
Hydrolized to AMP and PPi

23. Argininosuccinase
Cleaves argininosuccinate into arginine and fumarate.
Conserves carbon skeleton of aspartate in fumarate.
Cytoplasm

24. Arginase Hydrolyzes arginine into ornithine and urea.
Ornithine transported into mt
Urea excreted.

25. Urea Cycle is Linked to Gluconeogenesis
Aspartate and Fumarate link Urea Cycle to Gluconeogenesis.

26. 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???

27. 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.

28. 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

29. 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.

30. Fates of Carbon Skeletons

31. Amino Acids GLUCOGENIC BOTH KETOGENIC 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)
ESSENTIAL
(Val and His Three Methods)
Histidine (H, His)*
Methionine (M, Met)
Threonin (T, Thr)
Valine (V, Val)
BOTH KETOGENIC
NONESSENTIAL NONESSENTAIL
Tyrosine (Y, Tyr)
ESSENTIAL ESSENTAIL
(Iley Trpped BOTH Phesants) KETONES in Leu of Lysine
Isoleucine (I, ile) --Leucine (L, Leu)
Phenylalanine (F, Phe) -- Lysine (K, Lys)
Tryptophane (W, Trp)
Essential: TV FILM HW(R)K
Updated 2014

32. Amino Acids GLUCOGENIC BOTH KETOGENIC NONESSENTIAL A R * N D C E Q G P
(Val and His Three Methods)
H, His*
M, Met
T, Thr
V, Val
BOTH KETOGENIC
NONESSENTIAL NONESSENTAIL Y
ESSENTIAL ESSENTAIL
(Iley Trpped BOTH Phesants) KETONES in Leu of Lysine
I, Ile --L, Leu
F, Phe --K, Lys
W, Trp
Essential: TV FILM HW(R)K
Updated 2014

33. Definitions Glucogenic Amino Acids Ketogenic 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.

34. Pyruvate Amino Acids with 3 C backbone Ala, Trp Ser, Gly Cys Thr
Sulfur is converted to H2S, SCN- or SO32-
Thr
2-amino-3-ketobutyrate intermediate

35. Acetyl CoA Acetoacetyl CoA
Branched chain (Leu, Ile, Lys,) and aromatic amino acids (Phe, Trp, Tyr)

36. 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

37. 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

38. Phe / Tyr 1.Transamination 2. p-hydroxyphenylpyruvate hydroxylase
Dioxygenase
both atom of oxygen are incorporated into the product
3. Homogentisate oxidase
4.Isomerization
5. Hydrolyzed 2 3 1 4 5

39. Trp
Converted into alanine and acetoacetate via the action of several oxidases. 1 2 3 4

40. α-Ketoglutarate Amino acids with C5 skeletons Glu Gln Pro Arg His
Deaminated into α-Ketoglutarate
Gln
Hydrolyzed to glutamate by Glutaminase
Pro
Converted to glutamate γ-semialdehyde
Arg
His
Multistep process with 4-imidazolone 5-propionate intermediate

41. 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

42. Met degradation

43. Fumarate
Asp
Tyr
Some carbons
Phe

44. Oxaloacetate Asp Asn Direct deamination into Oxa
NH4+ removed by Asparaginase to form Asp which is deaminated

45. 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

46. Maple Syrup Urine Disease (branched-chain ketoaciduria)
Defective / missing branched-chain 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.

47. 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.

48. Other Diseases involving Enzymes of Amino Acid Degradation