protein turnover and amino acid catabolism

what is protein digestion and turnover?

• amino acids are obtained from the diet when proteins are digested

• cellular proteins are degraded to amino acids because of damage, misfolding, or changing metabolic demands

• excess amino acids cannot be stored or directly excreted, so they must be used for protein synthesis or degraded to ammonia

• dietary proteins are degraded to amino acids, which are absorbed by the intestine and transported into the blood

what are essential amino acids?

• amino acids that cannot be synthesized and must be acquired in the diet
• histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine

what is protein digestion?

• begins in the stomach, where the acidic environment denatures proteins into random coils, and is completed inthe intestine
• pepsin: primary proteolytic enzyme of the stomach; maximally active at pH 2
• partly digested proteins move from the stomach to the beginning of the small intestine (duodenum), stimulating the secretion of sodium bicarbonate and proteolytic enzymes from the pancreas
• aminopeptidases in the plasma membrane of intestinal cells enhance digestion

what are the products of protein digestion?

• absorbed by the small intestine
• free amino acids, dipeptides, and tripeptides are transported into the intestinal cells
• at least 7 different transporters exist, each specific to a different group of amino acids
• absorbed amino acids are released into the blood by a number of Na+-amino acid antiporters

what is protein turnover?

• the degradation and resynthesis of proteins
• cellular proteins are degraded at different rates
• takes place constantly in cells
• essential for removing short-lived or damaged proteins
• tightly regulated

what are the half-lives of proteins?

• range over several orders of magnitude
• ornithin decarboxylase, which catalyzes the synthesis of polyamines: ~11 minutes
• Hb: the life of the RBC
• lens protein, crystallin: life of the organism

what is ubiquitin?

• a small (76 aa), compact protein that tags proteins for destruction
• has 7 lysine residues and an extended carboxyl terminus, which is activated and linked to proteins targeted for destruction
• present in all eukaryotic cells
• highly conserved

what is the attachment of ubiquitin?

• attaches by its carboxyl-terminal Gly residue to the ε-amino groups of 1+ Lys residues on the target protein
• requires ATP hydrolysis
• forms an isopeptide bond because ε- rather than ⍺-amino groups are targeted
• 3 enzymes participate: ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), ubiquitin-protein ligase (E3)

what is ubiquitin-activating enzyme (E1)?

• adenylates ubiquitin and transfers it to a sulfhydryl group of Cys residue of E1
• requires ATP

what is ubiquitin-conjugating enzyme (E2)?

transfers ubiquitin to one of its own sulfhydryl groups

what is ubiquitin-protein ligase (E3)?

• transfers ubiquitin from E2 to an ε-amino group on the target protein
• brings E2 and the target protein together
• ubiquitin can be transferred directly or passed to a Cys residue of E3 first

what is the formation of chains of ubiquitin molecules?

• multiple ubiquitin molecules are typically added to a single protein substrate
• E3 can remain bound to the target protein and generate a chain of ubiquitin molecules
• E3 can dissociate after the first ubiquitin addition, then a chain can be extended by another E2/E3 pair
• ubiquitin can be added onto any of the seven 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

what are E3 ubiquitin ligases?

• provide the protein target specificity

in humans:

• 2 E1 ligase genes

• 30-50 E2 ligase genes

• over 600 E3 ligase genes

what is a tetraubiquitin chain?

• four ubiquitin molecules are linked by isopeptide bonds

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

• this unit is the primary signal for degradation when linked to a target protein

what is the importance of E3 proteins to normal cell function?

• proteins that are not degraded because of a defective E3 may accumulate, causing a disease of protein aggregation
• angelman syndrome: severe neurological disorder caused by a defect in a member of the E3 family
• overexpression of an E3 ligase (UBE3A): strongly linked to certain forms of autism
• inappropriate protein turnover can lead to cancer: several tumor suppressors, including BRCA1, are E3 ligases

what are the additional roles of ubiquitination?

also regulates proteins involved in:

• DNA repair
• chromatin remodeling
• innate immunity
• membrane trafficking
• autophagy

what are proteasomes (26S proteasome)?

• a large, ATP-driven protease complex that digests ubiquinated proteins
• complex of 2 compounds: one 20S catalytic unit arranged as a barrel; two 19S regulatory units that control access to the interior of the 20S catalytic subunit

what are the functions of the 19S regulatory unit?

• contains ubiquitin receptors that bind specifically to polyubiquitin chains
• uses ATP to unfold polyubiquinated chains and direct them into the catalytic core
• contains an isopeptidase that cleaves off intact ubiquitin molecules so that they can be reused

what are the components of the 19S complex?

six ATPases of the AAA+ class: a class of chaperone-like ATPases associated with the assembly, operation, and disassembly of protein complexes

what is the structure of the 20S proteasome?

• barrel-shaped and made up of 28 homologous subunits
• the subunits (⍺-, β-type) are arranged in 4 rings of 7 subunits each
• some of the β-type subunits include protease active sites at their amino termini

what are the proteolytic active sites of the 20S barrel?

3 types, each with a 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 hydroxyl group of the Thr residue attacks the carbonyl groups of peptide bonds, forming 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

what is the generation of free amino acids by the proteasome and other proteases?

• ubiquinated proteins are processed to peptide fragments
• ubiquitin is removed and recycled prior to protein degradation
• the peptide fragments are further digested to yield free amino acids, which can be used for biosynthetic reactions, most notably protein synthesis
• alternatively, the amino group can be removed and processed to urea and synthesize carbohydrates/fats, or used directly as a fuel for cellular respiration

what are processes regulated by protein degradation?

• via the ubiquitin-proteasome pathway
• e.g. gene transcription, cell-cycle progression, organ formation, circadian rhythms, inflammatory response, tumor suppression, cholesterol metabolism, antigen processing

what is bortezomib (velcade)?

• a dipeptidyl boronic acid inhibitor of the proteasome

• used as a therapy for multiple myeloma

• increases level of pro-apoptotic protein factors

what is HT1171?

• suicide inhibitor of the proteasome of M. tuberculosis
• has no effect on human proteasomes

what is the first step in amino acid degradation?

• removal of nitrogen
• the amino group is removed, and then the remaining C skeleton is metabolized to a glycolytic intermediate or to acetyl-CoA

what is amino acid degradation?

• amino acids not needed as building blocks are degraded to compounds able to enter the metabolic mainstream
• major site in mammals: liver
• muscles also readily degrade the branched-chain amino acids (Leu, Ile, and Val)

what is the oxidative deamination of glutamate?

• glutamate is oxidatively deaminated in the liver to yield ammonium ion (NH4+)
• the alpha-amino groups are converted into ammonium ions: transferred to ⍺-ketoglutarate, yielding glutamate

what is the role of aminotransferases?

• catalyze the transfer of an ⍺-amino group from an ⍺-amino acid to an ⍺-ketoacid

• reactions are reversible and can be used to synthesize amino acids from ⍺-ketoacids

what is aspartate aminotransferase?

• catalyzes the transfer of the amino group of aspartate to ⍺-ketoglutarate

aspartate + ⍺-ketoglutarate ↔ oxaloacetate + glutamate

what is alanine aminotransferase?

• catalyzes the transfer of the amino group of alanine to ketoglutarate

alanine + ⍺-ketoglutarate ↔ pyruvate + glutamate

what is the importance of blood levels of aminotransferases?

• serve a diagnostic function for liver damage

• the presence of alanine and aspartate aminotransferases in the blood is an indication of liver damage

• liver damage can occur due to: viral hepatitis, long-term excessive alcohol consumption, reaction to drugs (e.g. acetaminophen)

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

what do aminotransferases require?

the coenzyme, pyridoxal phosphate (PLP), a derivative of pyridoxine (vitamin B6)

what are the roles of pyridoxal phosphate enzymes?

• at the ⍺-C of amino acids, PLP-dependent enzymes catalyze decarboxylations, deaminations, racemizations, and aldol cleavages
• at the β-C and γ-C of amino acids, PLP-dependent enzymes catalyze elimination and replacement reactions

what is glutamate dehydrogenase?

• a mitochondrial enzyme that converts the N atom in glutamate to a free ammonia ion by oxidative deamination
• 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

what is the deamination of serine and threonine?

• serine dehydratase and threonine dehydratase directly deaminate their respective amino acids: PLP is the prosthetic group

• no transfer of the ⍺-amino group to ⍺-ketoglutarate is required

• dehydration precedes deamination

serine → pyruvate + NH4+
threonine → ⍺-ketobutyrate + NH4+

what is the fate of the ammonia ion?

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

• the sum of the reactions of aminotransferases and glutamate dehydrogenase is:

⍺-amino acid + NAD+ + H2O ↔ ⍺-ketoacid + NH4+ + NADH + H+

how is N from amino acids cleared?

through the urea cycle in the liver

how is N transported to the liver?

• by peripheral tissues as glutamine through glutamine synthetase to avoid high levels of circulating ammonia
• glutamine synthetase: catalyzes the synthesis of glutamine from glutamate and NH4+
• N of glutamine are eliminated by incorporation into urea in the liver

how do muscle cells transport N to the liver?

• as alanine by the glucose-alanine cycle
• during prolonged exercise and fasting, muscles use branched-chain amino acids as fuel
• the N removed is transferred (through glutamate) to alanine, which is released into the bloodstream
• in the liver, alanine is taken up and converted into pyruvate for the subsequent synthesis of glucose

what is the urea cycle?

• eliminates both N and C waste products
• 2 N atoms enter the cycle and leave as urea
• CO2 is simultaneously eliminated as it is hydrated to bicarbonate, which then enters the cycle

what is the first step of the urea cycle?

formation of carbamoyl phosphate, which then reacts with ornithine to begin the cycle

what is carbamoyl phosphate synthetase I?

• catalyzes the coupling of ammonia (NH3) with bicarbonate (HCO3-) to form carbamoyl phosphate: requires 2 molecules of ATP, making the reaction essentially irreversible
• occurs in the mitochondria
• mammals have 2 isozymes
• key regulatory enzyme for urea synthesis
• requires the allosteric regulator N-acetylglutamate for activity
• inhibited by acetylation and stimulated by deacetylation

what is N-acetylglutamate synthase?

• catalyzes the synthesis of N-acetylglutamate
• activated when amino acids are readily available

what is ornithine transcarbamoylase?

• catalyzes the transfer of the carbonyl group of carbonyl phosphate to ornithine, forming citrulline: transported into the cytoplasm
• occurs in the mitochondria

what is aspartate?

• condenses with citrulline

• the donor of the second N of urea

what is argininosuccinate synthetase?

• catalyzes the condensation of citrulline and aspartate to form argininosuccinate
• occurs in the cytoplasm
• requires ATP

what is argininosuccinase?

cleaves argininosuccinate into arginine and fumarate

what is the hydrolysis of arginine?

• arginase: hydrolyzes arginine to generate urea and ornithine
• ornithine is transported back into the mitochondria
• urea is excreted

how is the urea cycle linked to gluconeogenesis?

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

oxaloacetate can be:

• converted into glucose by gluconeogenesis

• transaminated to aspartate for another round of urea synthesis

how is N metabolism integrated with other metabolic pathways?

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

what are inherited defects of the urea cycle?

• causes hyperammonemia and can lead to brain damage

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

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