Biochemistry Ch 23 – Protein Degradation & Amino Acid Catabolism

Purposes of protein digestion

 Extracellular – digestion of dietary protein for nutritional purposes (most important dietary proteins
contain essential amino acids that we cannot synthesize)
o Use of amino acids for other protein synthesis
o For biosynthesis of nitrogenous compounds
o For fuel if energy is in dire need
 Intracellular – via self-compartmentalized proteases
o Control of amounts of specific proteins in the cell to control cell status (e.g., stages of cell
division) and metabolism
o Removal of denatured proteins (oxidized or partially unfolded) to prevent aggregation, as
dictated by various half-lives of different proteins
o Reuse of amino acids for synthesis or energy
 Specific purposes:
o Specific proteases for zymogen activation and post-translational modifications
o Immunoproteasomes for phago/endocytosis of foreign antigens

Digestion of dietary protein

 Starts in stomach
o Acidic pH  2 maintained via K/H pump (p-type ATPase)
o Pepsin: primary proteolytic enzyme (maximally active at pH 2), is nonspecific and can denature
most proteins
 Continues in lumen of intestine
o Pancreas secretes proteases as zymogens which are converted to active enzymes in lumen
o This digestion is enhanced by aminopeptidases anchored to extracellular plasma membrane of
intestinal cells; these will chop aa’s off N terminus of peptides
o Results in single amino acids and some tri-/dipeptides, which are transported into the
intestinal cells
o In cytosol of the intestinal cells, there is a peptidase to breakdown the tri-/dipeptides into
single aa’s
o The amino acids are released into the bloodstream to be absorbed by other tissues to be used
for biosynthesis or fuel

Proteins can be targeted for degradation

 Ubiquitin – small, highly conserved protein present in all eukaryotes that serves as signal for fate of
cellular proteins (i.e., a tag that marks proteins for destruction)
 Proteasome – general protease that acts on ubiquitin-tagged intracellular proteins (in the cytosol and
on the membrane facing the cytosol)

Mechanism of Ubiquitin-target protein attachment

 C-terminus of ubiquitin is attached to terminal amino group (ε-NH 2) of a lysine on the target protein
via an isopeptide bond
 A three-enzyme system (E1, E2, E3) targets & catalyzes the attachment of the first Ub:
o Ub is activated by ATP-dependent conjugation to E1 via thioester bond
o Ub is then transferred to E2
o E3 recognizes target protein and transfers Ub from E2 to lysine on target protein (accordingly
there must be a large family of E3 proteins in order to recognize all the targets)
 Biochemistry Ch 23 – Protein Degradation & Amino Acid Catabolism

(1) Activation of Ub by E1, at expense of ATP hydrolysis

(2) Transfer of Ub to E1
(3) Transfer of Ub from E1 to E2
(4a) E3 recognizes target protein, binds it and E2-Ub
(4b) E3 catalyzes transfer of Ub from E2 to lysine on target

 This initial ubiquitin will serve as point of attachment for a string of ubiquitins attached to each other
by isopeptide bonds (a series of 4 isopeptide bonds is the minimum to be targeted to proteasome)
 Different connections among the ubiquitin chain will target the protein to different locations
 Targeting signals unclear
o Half-life of a protein is largely determined by the aa on the N-terminus, so certain aa’s may
promote binding to ubiquitin sooner than other aa’s
o Certain aa sequences (e.g., PEST: Pro-Glu-Ser-Thr) may mark proteins for destruction
o Unknown for denatured proteins bc not all denatured proteins are tagged with Ub

Digestion of cytosolic proteins by proteasome – ubiquitin is the mark of death, proteasome is the executioner
 Proteasome – general (nonspecific) cellular protease
o large multi-unit assembly with 3 different active sites
o self-compartmentalized protease whose structure limits access of cellular proteins to active
sites (it would be dangerous to have it “open” in the cytosol, eating up proteins willy-nilly)
o 20S catalytic core is a sealed barrel composed of inactive α subunits and active β subunits
o Access to inside of the 20S barrel is controlled by 19S cap complexes:
 19S recognizes only polyubiquinated proteins for degradation
 19S removes the ubiquitins and releases them to be recycled
 19S unfolds the target protein (driven by ATP hydrolysis) and feeds it into the
proteolytic cavity of the 20S barrel, where it gets chopped into 7-10 aa peptides

Complete digestion of cellular proteins

 The resulting 7-10 aa peptides are broken down to single aa’s by
other compartmentalized peptidases
 These aa’s will be used for protein synthesis, or the amino group can
be removed and processed to urea and the carbon skeleton can be used to synthesize carbohydrates
or fats, or can be used directly as a fuel for cellular respiration
 There is no storage of single amino acids (except for in milk as protein source for infant)
 Proteasomes can also serve to digest foreign proteins for presentation by MHC Class I
 Biochemistry Ch 23 – Protein Degradation & Amino Acid Catabolism
 Processes regulated by protein degradation: gene transcription, progression through cell-cycle,
inflammatory response, cholesterol metabolism, antigen processing
Fate of amino acids released upon protein digestion?
First they will be used as building blocks for biosynthetic rxns. Any not needed for building blocks are
degraded to compounds that are able to enter the metabolic mainstream:
Amino acid catabolism (occurs mostly in liver)
 Step 1 – Removal of nitrogen from amino acids
o Transfer of amino group to α-ketoglutarate; resulting in an α-keto acid (carbon skeleton of aa
– no N is present) and glutamate
o This amino group transfer is catalyzed by aminotransferase (a.k.a., transaminase), specific for
each amino acid:
 e.g., Asp + α-ketoglutarate  oxaloacetate + glutamate (via aspartate aminotransferase)
 e.g., Ala + α-ketoglutarate  pyruvate + glutamate (via alanine aminotransferase)
o Pyridoxal phosphate (PLP)
 pyridoxene (vit B6) derivative that is a critical cofactor in all aminotransferases
 Remains tightly (noncovalently) bound to enzyme via Schiff-base linkage to a lysine
terminal (ε) amino group
 The α-amino group on the aa displaces the ε-amino group of the active-site lysine
residue, allowing transamination and many other reactions
 In Gogol’s words: “PLP is useful in amino reactions, to hold it in place so the enzyme can
do things with it” … I’m thinking we don’t need to know much more than that.
 Step 2 – Release of amine from glutamate
o The nitrogen atom in glutamate is converted into free ammonium (NH 4+) by oxidative
deamination, regenerating α-ketoglutarate
o This oxidative deamination is carried out by glutamate dehydrogenase, which uses NAD+
o Glutamate dehydrogenase is located in the mitochondria, so the toxic free ammonium is not
free in the cytosol
Aminotransferase Glutamate dehydrogenase

 Serine and threonine can be directly deaminated

o The α-amino groups of serine and threonine can be directly converted into NH 4+ without
first being transferred to an α-ketoglutarate
o This is permitted by the presence of a hydroxyl group on the aa’s
o These direct deaminations are catalyzed by dehydratases specific for serine and
threonine, which also are located in the mitochondria to sequester the NH 4+
o The mechanism involves a dehydration of OH group, followed by hydration/deamination
 Serine  Pyruvate + NH4+
 Threonine  α-ketoglutarate + NH4+

Nitrogen from muscle transported to liver

 Although most aa degradation occurs in liver, other tissues can degrade aa’s; muscle uses branched-
chain aa’s (Leu, Ile, Val) as a source of fuel during prolonged exercise
 Branched chain aa processing produces NH 4+ that cannot be processed so muscle transfers the NH 4+
to pyruvate, producing alanine which is released into the bloodstream
 Liver picks up the alanine and converts it back to pyruvate by transamination. The amino group, in
the form of glutamate, will be converted to urea. The pyruvate can be used for gluconeogenesis.
 Biochemistry Ch 23 – Protein Degradation & Amino Acid Catabolism
 If muscle is breaking down aa’s that means it must be hungry for energy, so in this glucose-alanine
cycle, liver will produce glucose to feed energy requirements.

Summary of glucose-alanine cycle:

During prolonged exercise & fasting, muscle
resorts to branched-chain aa’s as fuel. The N
removed is transferred through glutamate to
alanine, which is released in the
bloodstream and picked up by the liver
where it is converted to pyruvate for
synthesis of glucose.

Urea Cycle
 Excess NH4+ (that is not used for biosynthesis of nitrogenous compounds) is converted to urea via the
Urea Cycle in the liver – the urea will then be excreted
 Step 1 – Formation of carbamoyl phosphate
o Coupling of NH4+ with bicarbonate HCO3- to form carbamoyl phosphate
o This reaction is catalyzed by carbamoyl phosphate synthetase (synthetase indicates the
enzyme requires ATP) and occurs in the mitochondria
o The consumption of 2 ATP makes the synthesis of carbamoyl phosphate essentially irreversible
 Step 2 – Carbamoyl phosphate transferred to ornithine
o Still in liver mitochondria, carbamoyl phosphate is transferred to ornithine (aa but not used for
protein synthesis) to make citrulline, with a loss of phosphate
o Citrulline is transported out of mitochondria to continue the urea cycle
 Remaining steps to produce urea
o In cytosol, citrulline condenses with aspartate, resulting in arginosuccinate
o Arginosuccinate is lysed to arginine and fumarate
o Arginine is hydrolyzed to make urea and ornithine (ornithine is transported to mito for
subsequent cycles)
o Urea accumulates in cytosol and is secreted into bloodstream, where the kidneys filter it out
and into the urine

 Overall urea synthesis:

CO2 + NH4+ + 3 ATP + aspartate + H2O  urea + fumarate + 2 ADP + AMP + 4 P i
*Equivalent of 4 ATP are consumed to synthesize one molecule of urea!

Recycling fumarate to make aspartate

 Synthesis of fumarate by urea cycle is important bc it is a precursor for glucose synthesis
 Fumarate is hydrated & oxidized to form oxaloacetate
 Biochemistry Ch 23 – Protein Degradation & Amino Acid Catabolism
 Oxaloacetate can be used for gluconeogenesis or can be transaminated by another amino acid to
make aspartate
 So the point is: the urea cycle, gluconeogenesis, and the transamination of oxaloacetate are linked by
fumarate and aspartate

Processing the aa carbon skeleton

 So back to the first step of aa catabolism, where we transfer an amino group to form an α-ketoacid
and glutamate… ever since then we talked about what happens to the amine of glutamate (ammonium
 urea) What about the α-ketoacid, the carbon skeleton of the amino acid?
 Carbon skeletons are converted to intermediates in TCA cycle and/or gluconeogenesis pathway
o Glucogenic aa’s - amino acids that convert to intermediates to enter gluconeogenesis
o Ketogenic aa’s – amino acids that convert to acetyl/acetoacetyl CoA to produce ketone bodies
o Most aa’s are both types but mostly make glucose – only Leu & Lys are purely ketogenic
 Most common pathways:
o 3C aa’s  pyruvate
o 5C aa’s  glutamate  α-ketoglutarate For gluconeogenic pathway
o Nonpolar aa’s  succinyl CoA
o Branched chain aa’s  acetyl CoA
 acetoacetate (for ketone bodies), or propionly CoA (for FA generation)
o Aromatic aa’s require dioxygenases to break aromatic rings with O 2

Metabolic diseases of amino acid metabolism

 Hyperammonia – failures in the urea cycle that result in elevated NH 4+ which is toxic due to high levels
of Glu & Gln which results in changes in osmotic pressure
 Failures to process individual aa carbon skeletons results in accumulation of aa or α-ketoacid products:
o Maple syrup urine disease – branched chain α-ketoacids are not decarboxylated and
accumulate in the blood and urine (tx: limit BCAA intake)
o Phenylketonuria – failure to process phenylalanine to make tyrosine, results in accumulation
of phe (tx: severely restrict all sources of phe e.g., aspartame peptide used as artificial
sweetener)