lec 4 (mcbride) - protein turnover and AA catabolism

maintaining homeostasis…

requires metabolic regulation that coordinates the use of nutrient pools

protein digestion and turnover

• AA are obtained from diet when proteins are digested

• cellular proteins are degraded to AA b/c of damage, misfolding, or changing metabolic demands

• excess AA CANNOT be stored or excreted → must be used as metabolic fuel

protein production and consumption rates…

are controlled to maintain physiological levels and function

• homeostatic mechanisms adjust these rates to achieve production = consumption to maintain a physiological concentration required for life

proteins are degraded to…

amino acids

• dietary proteins are degraded to AA which are absorbed by the intestine and transported in the blood

• essential AA = AA that CANNOT be synthesized and must be acquired in the diet

essential AA

in humans CANNOT be synthesized from other dietary precursors

• histidine

• isoleucine

• leucine

• lysine

• methionine

• phenylalanine

• threonine

• tyrptophan

• valine

digestion of dietary proteins

begins in the stomach and is completed in the intestine

• protein digestion begins in the stomach where the acidic environment denatures proteins into random coils

• pepsin = the primary proteolytic enyzme of the stomach

  • max active at pH 2

• partly digested proteins move from the stomach → beginning of the small intestine (duodenum) → stimulating secretion of sodium bicarbonate and proteolytic enzymes from the pancreas

• aminopeptidases in the plasma membrane of intestinal cells enhance digestion

products of protein digestion…

are absorbed by the small intestine

• free AA, dipeptides, and tripeptides are transported into the intestinal cells

• at least 7 different transporters exist, each specific to a different group of AA

• absorbed AA are released into the blood by a number of Na+-AA antiporters

cellular proteins are degraded at…

different rates

• protein turnover = the degradation and resynthesis of proteins

  • takes place constantly in cells

  • essential for removing short-lived or damaged proteins

• the half-lives of proteins range over several orders of magnitude

  • ornithine decarboxylase, which catalyzes the synthesis of poly amines, is ~11 mins

  • hemoglobin = life of RBC

  • lens protein crystallin = life of organism

protein turnover

is tightly regulated

• ubiquitin = a small (76 aa) that tags proteins for destruction

  • present in all eukaryotic cells

  • highly conserved

• ubiquitin attaches by the carboxyl-terminal Gly residue to the ε-amino groups of +1 lys residues on target protein

  • requires ATP hydrolysis

  • forms an isopeptide bond because ε rather than α-amino groups are targeted 

ubiquitin

is a small, compact protein with 7 lysine residues

• has an extended carboxyl terminus which is activated and linked to proteins targeted for destruction

3 enzymes participate in the attachment of ubiquitin to protein

• ubiquitin-activating enzyme (E1) = adenylates ubiquitin and transfers it to a sulfhydryl group of a Cys residue of E1

• ubiquitin-conjugating enzyme (E2) = transfer ubiquitin to one of its own sulfhydryl groups

• ubiquitin-protein ligase (E3) = transfers ubiquitin from E2 to an ε-amino group of target protein

  • brings E2 and target protein together

  • ubiquitin be transffered directly or be passed to a cys residue of E3 first

multiple ubiquitin molecules are typically…

added to a single protein substrate

• E3 can remain bound to target protein and generate a chain of ubiquitin molecules

• E3 can dissociate after the first ubiquitin addition and a chain can be extended by another E2/E3 pair

• ubiquitin can be added onto any of the 7 Lys or the N-terminus of the previous ubiquitin

• a chain of 4+ ubiquitin molecules linked via Lys 48 = especially effective signal for protein degradation

E3 ubiquitin ligases provide…

the protein target specificity

• in humans:

  • 2 E1 ligase genes

  • 30-50 E2 ligase genes

  • over 600 E3 ligase genes

in a tetraubiquitin chain…

4 ubiquitin molecules are linked by isopeptide bonds

• the ε-amino group of a Lys residue of 1 ubiquitin is linked to the terminal carboxylate of another

• this unit = primary signal for degradation when linked to target protein

degrons

are amino acid sequences that control protein half lifew

• degron = specific sequence of AA that indicate a protein should be degraded

• for many proteins, the amino-terminal residue AA (N-degron) = important degradation signal for E3 enzyme

  • may only be exposed after proteolytic cleavage

  • may be added after protein synthesis

  • may require other modifications

• other degrons include cyclin destruction boxes and PEST sequences

cytoplasmic yeast

dependence of the half-lives of cytoplasmic yeast proteins on the identity of their amino-terminal residues

• highly stabilizing residues (half life > 20 hours)

  • ala

  • cys

  • gly

  • met

  • pro

  • ser

  • thr

  • val

• intrinsically destabilizing residues (half life = 2-30 mins)

  • arg

  • his

  • lie

  • leu

  • lys

  • phe

  • trp

  • tyr

• destabilizing resides after chemical modification (half life = 3-30 mins)

  • asn

  • asp

  • gln

  • glu

importance of E3 proteins to normal cell function

• proteins that are NOT degraded b/c of a defective E3 may accumulate → causing a disease of protein aggregation

• angelman syndrome = severe neurological disorder characterized by an unusually happy disposition, cognitive disability, absence of speech, uncoordinated movement, and hyperactivity

  • caused a defect in a member of E3 family

• overexpression of E3 ligase causes autism

• inappropriate protein turnover → cancer

additional roles of ubiquitination

• ubiquitination also regulates proteins involved in:

  • DNA repair

  • chromatin remodeling

  • innate immunity

  • membrane trafficking

  • autophagy

the proteasome

digests the ubiquitin-tagged proteins

• proteasome (26S proteasome) = a large, ATP-driven protease complex that digests ubiquitinated proteins

• the 26S proteasome = complex of 2 components

  • one 20S catalytic unit arranged as barrel

  • two 19S regulatory units that control access to the interior of the 20S catalytic subunit

functions of the 19S regulatory unit

• the 19S regulatory units:

  • contain ubiquitin receptors that bind specifically to polyubiquitin chains

  • uses ATP to unfold polyubiquitinated chains and direct them into catalytic core

  • contains an isopeptidase that cleaves off intact ubiquitin molecules so they can be reused

• key components of the 19S complex = 7 ATPases of the AAA+ class

  • a class of chaperone-like ATPases associated with:

    • assembly

    • operation

    • and disassembly of protein complexes

20S proteasome

is barrel-shaped and made up of 28 homologous subunits

• the subunits (α-type, red; β-type, blue) are arranged in 4 rings of 7 subunits each

• some of the β-type subunits (right) include protease active sites at their amino termini

proteolytic active sites of the 20S barrel

• there are 3 types of active sites in the β subunit, each with different specificity

  • chymotrypsin-like: cleaves after large hydrophobic amino acids

  • trypsin-like: cleaves after basic amino acids

  • caspase-like: cleaves after acidic amino acids

• all active sites employ an N-terminal Thr residue

  • the OH group of the Thr residue attacks the carbonyl groups of peptide bonds → forms acyl-enzyme intermediates

  • substrates are degraded in a processive manner WITHOUT intermediate release

  • substrates are reduced to peptides ranging from 7-9 residues before release

the proteasome and other proteases generate

free amino acids

• ubiquitinated proteins are processed to peptide fragments

• ubiquitin is removed and recycled prior to protein degradation

• peptide fragments are further digested to yield free AA which can be used for biosynthetic reactions; most notably protein synthesis

  • alternatively, the amino group can be removed and processes to urea and the carbon skeleton can be used to synthesize carbohydrate or fats or used directly as fuel for cellular respiration

processes regulated by protein degradation

• many biological processes are controlled, at least in part, by protein degradation via the ubiquitin-proteasome pathway

• processes regulated by protein degradation

  • gene transcription

  • cell-cycle progression

  • organ formation

  • inflammatory response

  • tumor suppression

  • cholesterol metabolism

  • antigen processing

protein degradation can be used to regulate…

biological function

• bortezomib (velcade) = dipeptidyl boronic acid inhibitor of the proteasome

  • used as therapy for multiple myeloma

• degrons = used as regulatory mechanisms for protein expression

• HT1171 = suicide inhibitor of the proteasome of M. tuberculosis

  • has NO effect on human proteasomes

first step in amino acid degradation

is the removal of nitrogen

• AA NOT needed as building blocks are degraded to compounds able to enter the metabolic mainstream

• amino group is removed and remaining carbon skeleton is metabolized to glycolytic intermediate or to acetyl CoA

• major site of AA degradation in mammals = liver

• muscles also readily degrade the branched-chain AAs (leu, Ile and Val)

alpha-amino groups

are converted into ammonium ions by oxidative deamination of glutamate in liver

• α-amino groups → α-ketoglutarate → yielding glutamate

• glutamate is oxidatively deaminated in the liver to yield ammonium ion (NH4+)

role of aminotransferases

• aminotransferases (transminases) = catalyze the transfer of α-amino group from an α-amino acid → α-ketoacid

  • reaction are reversible and can be used to synthesize amino acids from α-ketoacids

aspartate aminotransferase and alanine aminotransferase

• aspartate aminotransferase = catalyze the transfer of amino group of aspartate → α-ketoglutarate; end result = oxaloacetate + glutamate

• alanine aminotransferase = catalyze the transfer of the amino group of alanine → α-ketoglutarate; end result = pyruvate + glutamate

blood levels of aminotransferase serve as…

diagnostic function for liver damage

• the presence of alanine and aspartate aminotransferase in the blood = indication of liver damage

• liver damage can occur due to:

  • viral hepatitis

  • long-term excessive alcohol consumption

  • reaction to drugs

• in cases of liver damage, liver cell membranes are damaged and aminotransferases leak into blood

aminotransferases require…

pyridoxal phosphate (PLP) from vitamin B₆

• aminotransferases require the coenzyme pyridoxal phosphate (PLP) - a derivative of pyridoxine (vitamin B6)

transamination

• step 1

  • transfer of amino acid group from amino acid substrate → PLP and release of keto acid

  • example: for alanine aminotransferase, AA = alanine and released ketoacid is pyruvate

• step 2

  • transfer of amino acid from coenzyme → keto acid → new amino acid

  • example: for alanine aminotransferase, the ketoacid is α-ketoglutarate and the released AA is glutamate

pyridoxal phosphate enzymes

catalyze a wide array of reactions

• at the α-carbon of amino acids, PLP-dependent enzymes catalyze:

  • decarboxylations

  • deaminations

  • racemizations

  • aldol clevages

• at the β-carbon and γ-carbon of AA, PLP-dependent enzymes catalyze elimination and replacement rxn

role of glutamate dehydrogenase

• glutamate dehydrogenase = a mitochondrial enzyme that converts the nitrogen atom in glutamate → free ammonia ion by oxidative deamination

  • is essentially a liver-specific enzyme

  • can use either NAD+ or NADP+

  • proceeds by dehydrogenation of the C-N bond, followed by hydrolysis of the ketimine

  • allosterically inhibited by GTP and stimulated by ADP in mammals

serine and threonine can be…

directly deaminated

• serine dehydratase and threonine dehydratase directly deaminate their respective AA

  • PLP = prosthetic group (non-protein component that is tightly bound to protein and essential for biological function)

• NO transfer of the α-amino group to α-ketoglutarate is required

• dehydration precedes deamination

  • serine → pyruvate + NH4+

  • threonine → α-ketobutyrate + NH4+

fate of the ammonia ion

• in most terrestrial vertebrates, NH4+ converted into urea → excreted

• sum of the reactions of aminotransferases and glutamate dehydrogenase is → second equation in image

peripheral tissues transport…

nitrogen to the liver

• muscles use branched-chain AA as fuel during prolonged exercise and fasting

• muscles lack enzymes of the urea cycle

• nitrogen is transported from muscle → liver as alanine (through glutamate) in the glucose-alanine cycle

• glutamine synthetase = catalyzes the synthesis of glutamine from glutamate and NH4+

  • nitrogens of glutamine can be eliminated by incorporation into urea in the liver

pathway integration: the glucose-alanine cycle

allows muscle cells to use AA as fuel

• during prolonger exercise and fasting, muscles use branched-chain AA as fuel

• nitrogen removed is transferred (through glutamate) to alanine which is released into the blood stream

• in the liver, alanine is taken up and converted into pyruvate for the subsequent synthesis of glucose

urea cycle

eliminates both nitrogen and carbon waste products

• 2 nitrogen atoms enter the cycle and leave as urea

• carbon dioxide is simultaneously eliminated as it is hydrated to bicarbonate which then enters the cycle

urea cycle begins with…

formation of carbamoyl phosphate

• carbamoyl phosphate synthetase I = catalyzes the coupling of ammonia (NH3) with bicarbonate (HCO3-) → form carbamoyl phosphate

  • occurs in mitochondria

  • mammals have 2 isozymes

  • requires 2 molecules of ATP, making reaction essentially irreversible

carbamoyl phosphate synthetase I is…

the key regulatory enzyme for urea synthesis

• carbamoyl phosphate synthetase I:

  • requires the allosteric regulator N-acetylglutamate for activity

  • is inhibited by acetylation and stimulated by deactylation

• N-acetylglutamate synthase = catalyzes the synthesis of N-acectylglutamate

  • activated when AA are readily available

carbamoyl phosphate reacts with…

ornithine to begin urea cycle

• ornithine transcarbamoylase = catalyzes the transfer of the carbamoyl group of carbamoyl phosphate to ornithine forming citrulline

  • occurs in the mitochondria

• citrulline is transported into the cytoplasm

citrulline

condenses with aspartate

• aspartate is the donor of the second nitrogen of urea

• argininosuccinate synthetase = catalyzes the condensation of citrulline and aspartate → argininosuccinate

  • occurs in cytoplasm

  • requires ATP

cleavage of argininosuccinate

• argininosuccinase = cleaves argininosuccinate → arginine and fumarate

hydrolysis of arginine

• arginase = hydrolyzes arginine to generate urea and ornithine

• ornithine is transported back into mitochondria

• urea is excreted

urea cycle is linked to gluconeogenesis

• the stoichiometry of the urea cycle = photo

• fumarate is hydrated to malate which is in turn oxidized oxaloacetate

• oxaloacetate can be:

  • converted into glucose by gluconeogenesis

  • transaminated to aspartate for another round of urea synthesis

nitrogen metabolism is…

integrated with other metabolic pathways

• urea cycle, gluconeogenesis, and the transamination of oxaloacetate are linked by fumarate and aspartate

inherited defects of the urea cycle…

cause hyperammonemia and can lead to brain damage

• any defect in the urea cycle leads to an elevated level of NH4+ in the blood (hyperammonemia)

• high levels of NH4+ may:

  • inappropriately activate an Na+-K+-Cl- cotransporter, disrupting the osmotic balance of the nerve cell and causing cellular swelling

  • disrupt neurotransmitter systems

  • impact energy metabolism, levels of oxidative stress, nitric oxide synthesis and signal transduction pathways

argininosuccinase deficiency

can be managed by supplementing the diet with arginine

• argininosuccinate deficiency is treated by:

  • restricting total protein intake

  • supplementing the diet with arginine

• excess nitrogen is excreted in the form of argininosuccinate

both carbamoyl phosphate synthetase and ornithine transcarbamylase deficiencies…

can be treated with supplementation

• by supplementing the diet with benzoate and phenylacetate, excess nitrogen can be excreted in the form of hippurate and phenylacetylglutamine