Protein Turnover Flashcards
Where does Protein Turnover Take place?
1) Proteins from diet are hydrolyzed in the digestive tract
2) Proteins within each cell are broken within that cell in a proteasome
Degradation of Amino Acids requires:
Excess amino acids are neither stored nor excreted
Degradation:
1) Deamination-removal of amino group which enters the Urea Cycle
2) Conversion of carbon skeleton to Glucose/glycogen by converting to:
- acetyl CoA
- Acetoacetyl CoA
- Pyruvate
- Citric Acid Intermediate
Ubiquitin (Ub)
Protein found in all eukaryotes
“Mark of Death” tags protein for proteolysis
The Glycine C-Terminal of Ub covalently attaches to the R group of Lys on the protein
Ub forms polymers called polyubiquitination
Ub attachment to protein requires 3 enzymes before attachment:
1) E1 or Ubiquitin-activating enzyme
- activates Ub by Adenylation-attachment to AMP
- E1 subunit displaces AMP and Sulfhydryl binds to the Glycine C-terminal carboxylate forming a thioester bond
2) E2 or Ubiquintin-conjugating enzyme
- activated Ub transferred from Sulfhydryl of E1 to sulfhydryl of E2
3) E3 or Ubiquintin-protein ligase
- catalyzes the transfer of Ub from E2 to target protein
- largest gene family in humans
Structure of Proteasome
26S Large Protease Complex attacks ubiquinated proteins
1) Two 19S caps contains AAA class ATPase activity
-ATPase associated with various activities
2) One 20S Catalytic Core
4 Rings of 7 subunits-28 subunits total
-14 alpha (outer rings)-7 isoforms
-14 beta (inner rings)- 7 isoforms
Beta subunits contain the Proteolytic active sites
-Threonine residue acts as nucleophile to attach carbonyl of peptide bond
Action of Proteasome
-Function
Hydrolyzes Ubiquinated Proteins
Digest proteins to 7-9 amino acid peptides, which are released from proteasome and further degraded to amino acids by cellular proteases
Bortizomib
or Velcade
Inhibitor of Proteasome
-Therapy for multiple myeloma
What is the primary site of amino acid degradation?(removal of nitrogen)
Liver
-muscle is secondary site of amino acid degradation for branched chain aliphatic amino acids (L, I, V)
Nitrogen Removal from Amino Acids requires what enzymes?
Aminotransferase (AKA transaminases)
Glutamate dehydrogenase
Aminotransferase
AKA transaminases
-alpha amino group transfered to alpha ketogluterate to form glutamate
Ex: Aspartate Aminotransferase, Alanine Aminotransferase
-Reversible
-also used in synthesis of amino acids
-Prosthetic Group=PLP-pyridoxal Phosphate (Vit B6)
Glutamate Dehydrogenase
Converts nitrogen of glutamate to ketone to form alpha ketoglutarate and releases ammonium ion
- localized to the mt of liver
- Either uses NAD+ (Degradation) and NADP+(synthesizing)
Close to equilibrium in liver
-Direction of reaction depends on Substrate or Products concentration, but normally, driven forward by removal of Ammonium
PLP
Pyridoxal Phosphate (PLP) -prosthetic group
Function: Group transfer to or from amino acid
-electron acceptor/donor
Vit precursor=Vit B6-pyridoxine
Serine and Threonine degradation
Directly deaminated (Self deamination) -Amino group is not transferred to alpha keto glutarate
1) Serine dehydratase
- dehydrates and deaminates serine to produce pyruvate and ammonium
2) Threonine Dehydratase
- dehydrates and deaminated threonine to produce alpha-ketobutyrate and Ammonium
What are we going to do when the amino groups are released from muscles?
Peripheral tissues transport Nitrogen to the liver Glucose-Alanine Cycle
1) Muscles use branched amino acids as fuel and amino group is transferred through glutamate to alanine
2) Alanine with the amino group enters the blood and is transported to the liver
2) Once in the liver, transfers amino group to alpha ketoglutarate to form glutamate-> NH4+ through urea cycle
- Alanine is converted to pyruvate for gluconeogenesis to produce glucose
Urea Cycle
Cycle responsible for synthesis of urea
-Urea=form of nitrogen excreted in vertebrates
Carbamoyl Phosphate
- intermediate in urea cycle
- synthesized from NH3 and HCO-(Bicarbonate derived from hydration of CO2)
- Carbamoyl Group has a high phosphate transfer potential due to anhydride bond
How much urea does Human excrete per year?
10Kg=22lbs
Source of Atoms in Urea?
1 N from free ammonium (NH4+)
C from HCO3- (bicarbonate derived from hydration of CO2)
1 N from Aspartate
Carbamoyl Phosphate Synthetase
-reaction
Catalyzes the 3 Step synthesis of Carbamoyl Phosphate
- matrix of mitochondria
- uses 2 ATP to synthesize Carbamoyl Phosphate
- carbamoyl phosphate has a high phosphate transfer potential due to anhydride bond
1) Bicarbonate (HCO3-) phosphorylated by Phosphate from ATP forming Carboxyphosphate
2) Carboxyphosphate reacts with ammonia to form carbamic acid
3) Carbamic acid is phosphorylated by ATP to yield Carbamoyl Phosphate
Isozyme catalyzes the synthesis of carbamoyl phosphate for use in pyrimidine synthesis
Carbamoyl Phosphate Synthetase
-structure
1)Three Reaction sites
-Glutamine hydrolysis site
-Bicarbonate Phosphorylation site
-Carbamic Acid phosphorylation site
2) Substrate Channeling
-Substrate passed through channel from one active site to the next active site without releasing the enzyme
Benefits?
-increases rate of reaction
-protects libile substrates from degradation by hydrolysis
Ornithine Transcarbamoylase
Carbamoyl Phosphate + Ornithine-> Citrulline
- occurs in mt matrix
- following synthesis citrulline is transported to the cytoplasm
Arininosuccinate Synthetase
Citrulline + Aspartate-> Argininosuccinate
- condensation reaction at the expense of ATP which is hydrolyzed to AMP + PPi
- occurs in the cytoplasm
Arginosuccinase
Cleaves Argininosuccinate-> Arginine + Fumurate
- conserves carbon skeleton of aspartate in fumurate
- occurs in cytoplasm
Arginase
Arginine-> Ornithine + Urea
- Ornithine transported into mitochondria
- Urea excretes
What links Urea Cycle to Gluconeogenesis?
Aspartate and Fumarate
Ketogenic Amino Acids
Carbon skeletons are converted into intermediated (Acetyl CoA or Acetoacetyl CoA) that can form ketone bodies or Fatty Acids
-NOT substrates for Gluconeogenesis
Glucogenic Amino Acids Definition
Carbon Skeletons are converted into intermediates that can be used to synthesize glucose
What are the the intermediates that the Carbon Skeletons from deaminated Amino Acids are converted into?
7
- Pyruvate
- Acetyl CoA (KETOGENIC)
- AcetoAcetyl CoA (keotgenic)
- a-ketoglutarate
- Succinyl CoA
- Fumurate
- Oxaloacetate
Overview Mech of Urea Cycle:
1) Carbamoyl Phosphate + Ornithine (mt)-> Citrulline
2) Citrulline + Asparate-> Argininosuccinate
3) Arginino Succinate -> Fumurate + Arginine
4) Arginine + H2O -> Ornithine + Urea
Whats required for degradation of Aromatic Amino Acids
Oxygenase!
Phenylalanine Hydroxylase (Monooxygenase)
- Hydroxylates Phe-> Tyr
- Tetrahydrobiopertin=electron carrier
What are the 2 types of Oxygenases?
Monooxygenase
-each atom of O2 is incorporated into 2 different products
Dioxygenase
-both atoms of O2 are incorporated into single product
Tetrahydrobiopertin (BH4)
electron carrier derived from Biopertin
Synthesized in vivo from phenylalanine, not a vitamin bc it is synthesized in the body
1) Phenylalanine hydroxylase
- synthesized dihydrobiopertin by hydroxylating phenylalanine which forms Tyrsoine
2) Dihydrofolate reductase
- reduces dihydrobiopertin to tetrahydrobiopertin using NADPH
3) Dihydropteridine reductase
- reduces quinoid dihydrobiopertin using NADPH
Mechanism to breakdown Phe
1) Phe-> Tyr by deamination using Phenylalanine Hydroxylase with Tetrahydrobiopertin
2) Transamination–Tyrosine amino transferase
Tyr-> P-hydroxyphenylpyruvate
3) P-hydroxyphenylpyruvate hydroxylase
- Dioxygenase-both oxygen atoms incorporated to Product
- Homogensitate
3) Homogentisate oxidase (dioxygenase)
4) Isomerization
5) Hydrolyzed
- Fumarate + Acetoacetate (reason why they are both glucogenic and ketogenic)
Trp degradation mechanism
converted into alanine and Aspartate via the action of several oxidases
1)Dioxygenase, 2) NH3+ 3) Monoxygenase,4)alanine released 5) dioxygenase 6)Acetoacetate x11 steps
Amino Acids -> Alpha Ketogluterate
Gln-> Glu via Glutaminase
Glu deaminanted pinot Alpha Ketogluterate via Glutamate dehydrogenase NAD+
Arg-> Ornithine-> Gluatamate Y semialdehyde -> Glutamate
Pro -> Glutamate Y semiadlehdye-> Glutamate
His:
multistep process with 4-imidazolon 5-Propionate intermediate-> Glutamate
Amino Acids-> Succinyl CoA
Val/ Isoleucine-> Propionyl CoA-> Methylmalonyl CoA-> Succinyl CoA
Methionine-> SAM->S-adenosylhomocysteine-> Homocysteine + Serine(R group attaches and loses O bond to S)->Cystathionine-> release of cysteine + a-ketobutyrate-> Propinoyl CoA—»»»Sucicinyl CoA
Amino Acids-OAA
Asp
-direact deamination into OAA
Asn:
-NH4+ removed via Asparaginase to form Asp
Degradation of cellular proteins
Proteins turnover within the cell
- Damaged/misfolded are identified by Ub and destroyed quickly
- Proteins that are no longer needed
Half life of proteins vary from protein to protein
Deteremine by N-terminal residue
-R or L favors ubiquination=fast destruction
-M or P disfavors ubiuination=slow destruction/long half life
Excretion of Nitrogen:
-different species
Terrestrial vertebrates
-Ureotelic-excrete excess nitrogen as urea
Aquatic vertebrates and invertebrates
-ammoniotelic-excrete excess nitrogen as ammonium
Birds and reptiles
-Uricotelic-excrete excess nitrogen as uric acid
Tetrohydrobiopertin
BH4
-electron carrier
-synthesized in vivo from phenylalanine, not a vitamin because it is synthesized in the body
Phenylalanine hydroxylase-synthesizes Dihydrobiopertin by hydroxylating phenylalanine
dihydrobiopterin reductase
-reduces dihydrobiopterin to tetrahydropterin
Dihydropteriteridine reductase
-reduces quinoiod dihydrobiopterin to tetrahydropterin
Degradation of Dietary Proteins
Begins in stomach:
-ph=2, acidic environment allows the primary proteolytic enzyme, nonspecific, pepsin to function at maximum activity
-breaksdown proteins to Oligopeptides and amino acids
and head to the lumen of intestine
lumen of intetine
- Most enzymes involved in digestion are synthesized in the pancreas and secreted as Zymogens
- Aminopeptidase-nonspeicfic protease in the intestinal wall of cell that sequentially hydrolyzes proteins from the amino terminal end
- then amino acids enter blood stream
Hyperammonemia
inherited disease of urea cycle
-elevate levels of ammonium (NH4+) in blood
causes include:
- lack/reduced synthesis of carbamoyl phosphate
- lack/reduced activity of the four enzyme of urea synthesis
Treatments of Hyepammonemias
1) Argininosuccinase deficiency
- Provide excess arginine in diet
- restrict Total protein in diet which reduces the amount of nitrogen to be excreted
Xs supply of arginine is converted to ornithine which reacts with carbamoyl phosphate to form citrulline which reacts with aspirate to formargininosuccine which is excreted
2) Carbamoyl Phosphate synthetase or Ornithine transcarbamoylase deficiency treatment
- excess nitrogen accumulates in glycine and glutamine
- restrict protein in diet
Alcaptonuria
First description of an inborn error of metabolism
Defective degration of Phe to Tyr
- recessive inheritance
- homogentisate accumulates and is excreted in the urine as a highly colored polymer
- harmless condition
Maple Syrup Urine Disease
Branched-chain Ketoaciduria
Defective/missing branched-chain dehydrogenase
Defective degradation of Ile, Valine, Leucine
-causes mental and physical retardaiton
-detected by mass spec
PKU
Phenylketonuria
defective/missing phenylalanine hydroxylase(or the tetrahydrobiopterin cofactor)
-Autosomal recessive
-Untreated PKU can caused reduced life expectancy 20s-30s and retardation
