Chapter 23:Protein Turnover and

Amino Acid Catabolism

Copyright © 2007 by W. H. Freeman and Company

Berg • Tymoczko • Stryer

BiochemistrySixth Edition

Amino Acid Metabolism

Liver is the primary site for amino acid metabolism.

Each amino acid has a pathway for catabolism and separate one for anabolism. Actually the pathways differ in different organisms.

For mammals: Essential amino acids must be obtained from diet.Nonessential amino acids - can be synthesized .

Exogenous Protein Digestion

In stomach and intestinal

Most dietary protein is hydrolyzed to amino acids and small peptides in the stomach and intestine.

Digestive Proteases

Stomach (pH ~ 1-2): Pepsin, Gastricin, Chymosin

Intestine (pH ~ 8): Trypsin, Chymotrypsin,

Carboxypeptidase, Elastase Enteropeptidase, Aminopeptidases

Endogenous protein is also catabolized but by a method that differs from that above. Also, endogenous proteins have varying lifetimes.

Endogenoous Protein Turnover

Proteins exhibit continuous turnover (synthesis and degradation). Protein half-lives are from minutes to months, most are short. The amino terminal residue is a factor in selection of some protein.

ornithine decarboxylase t½ ~11 min liver & plasma protein ~2-10 days muscle protein ~180 days collagen ~1000 days

In eucaryotes, some proteins are targeted for degradation by a covalent attachment through lysine residues of the target protein to the C- terminus of ubiquitin, a small 76 residue peptide.

Ubiquitin on Lysine

Attachment of a lysine in the target protein to the C-term glycine of ubiquitin via an isopeptide bond.

The signal for protein death.

Ubiquitin

Attachment of ubiquitin to Lys requires three enzymes:

E1. Ubiquitin-activating enzyme (uses ATP)

E2. Ubiquitin-conjugating enzyme (assembles Ubiq., the target protein and E3).

E3. Ubiquitin-protein ligase (forms the Gly-Lys bond).

Mechanism of Attachmentan acyladenylate

Many isoforms of E3 exist. These select proteins for degradation.

Ubiquitin Chains

Sequential attachment of C-term Gly to Lys48 of another ubiquitin forms tetra-ubiquitin. This extended structure serves as an enhanced degradation signal.

A ProteasomeA Proteasome is a large ATP dependent complex that hydrolyzes the ubiquitinated proteins.

Degradation Events

The core of subunits contain the active sites and all have an N-term Thr.

The subunits on the ends serve as regulatory caps that block access to the active sites.

Procaryotic vs Eucaryotic

Procaryotes have a proteasome analog of that in eucaryotes but the function is unclear since ubiquitin has not been found. In procaryotes, all α subunits are identical and all β subunits identical whereas in eucaryotes these subunits exhibit a number of isoforms.

Procaryotes do have a ubiquitin-like protein but it is is used in the synthesis of thiamine and not protein degradation.

Procaryotic vs Eucaryotic

Procaryotic vs EucaryoticFor thiamine synthesis For protein degradation

Amino Acid Catabolism

First step amino acid catabolism is generally removal of the -amino group.

Carbon chains are then altered for entry into central pathways of carbon metabolism.

The amino acids from either degraded proteins or from a dietary source can be used for the biosynthesis of new proteins.

During starvation proteins are degraded to amino acids to support glucose formation.

Methods for Removal of NH3

1. Transamination:

amino acid + -ketoglutarate -ketoacid + Glu

2. Glutamate dehydrogenase:

Glu + NAD+ -ketoglutarate + NADH + NH4+

3. Direct deamination:

Ser pyruvate

His urocanate (resonance driven)

4. Amide hydrolase

Gln or Asn Glu or Asp + NH4+

1. Transamination

A pyridoxal phosphate (PLP) mediated reaction. -ketoglutarate, the normal -keto acid used, forms glutamate.

PLPThe aldehyde group forms a schiff base with a Lys on a transaminase enzyme.

PLP enzymes are typically involved in: transamination, decarboxylation, or racemization.

Transaminase Mechanism

Lysyl linkage is displaced by an amino acid.

Transaminase MechanismLoss of a proton and formation of a different Schiff base.

Transaminase Mechanism

Reprotonation

Transaminase MechanismSchiff base hydrolysis removes the amino group and gives an -ketoacid. Bringing in -ketoglutarate and reversing these steps gives Glu.

Asp AminotransferaseAmino acid not shown, but Arg binds with –COO-

Bond Cleavage

2. Glutamate Dehydrogenase

The requirement for NAD+ or NADP+ in this enzyme varies. Glu (from transamination) -- > -ketoglutarate

3. Direct Deamination

1. Serine Dehydratase (uses PLP in Ecoli).

2. In other amino acids direct deamination is driven by extended conjugation.

4. Amide Hydrolysis

NH4+ is removed from asparagine and

glutamine by the enzymes asparaginase and glutaminase.

asparaginase asparagine ------------ > aspartate + NH4

+

glutaminase glutamine ---------- > glutamate + NH4

+

Disposition of NH4+

The ammonium ion (which is toxic) formed by action of a transaminase and glutamate dehydrogenase (below) or other reactions, goes to the urea cycle (in the liver).

Transaminase Glutamate DH

Transport of NH4+

NH4+ is transported to the liver by either of two

methods.

1. Glucose-Alanine Cycle (next slide)

2. Glutamine

glutamine NH4

+ + glutamate + ATP ------- > glutamine + ADP + Pi synthetase

glutaminase glutamine ---------- > glutamate + NH4

+

Glucose-Alanine Cycle

NH4+ to Urea

Waste nitrogen must be removed (a high conc. of ammonia is cytotoxic)Fish and many aquatic organisms excrete NH4

+, Terrestrial vertebrates synthesize urea, Birds, reptiles synthesize uric acid.

The liver processes NH4+ into urea using carbamoyl-

phosphate synthetase I and enzymes of the urea cycle.

Incorporation of NH4+

Requires carbamoyl phosphate synthetase.

Carbamoyl phosphate synthetase I catalyzes removal of ammonia using the energy from ATP to form carbamoyl phosphate. This is the normal feeder for the urea cycle which is a liver pathway.

Carbamoyl phosphate synthetase II uses glutamine as a source of ammonia again using the energy from ATP to form carbamoyl phosphate. This provides carbamoyl-P for pyrimidine synthesis.

Carbamoylphosphate Synthetase I

A mitochondrial enzyme, requires 2 ATP

Urea Cycle

Four reactions.

One in the mitochondria.

Three in the cytosol.

Urea Cycle, Reaction 1

Transfer of the carbamoyl group in the mitochondria.

Urea Cycle, Reaction 2A citrulline:ornithine antiport moves citrulline to the cytosol. The second NH2 for urea comes from Asp. An adenylated citrulline intermediate gives PPi.

Urea Cycle, Reaction 3

Cleavage of fumarate, production of arginine

Urea Cycle, Reaction 4Cleavage of urea from arginine gives urea. The ornithine is ready to begin a new cycle.

Recycling to AspartateMalate dehydrogenase occurs in mitochondria and in the cytosol.

N-Acetylglutamate

An activator of CPS I which provides substrate for the urea cycle.

Glu is a product of Gln hydrolysis by CPS II in pyrimidine synth.

An intermediate in ornithine synthesis.

N-AcGlu is synthesized when NH4+ levels increase

during amino acid catabolism.

Structural similarity

Blue - Ornithine transcarbamoylase from the urea cycle

Red - Aspartate transcarbamoylase from pyrimidine synthesis

Functional similarity

Transfer of an amino group by incorporation of aspartate and elimination of fumarate occurs in both the urea cycle and pyrimidine synthesis.

AA Carbon Skeletons

In catabolism of their carbon skeletons, amino acids are referred to as being either glucogenic or ketogenic depending upon the structures of the degradation products.

Glucogenic amino acids degrade to pyruvate or citric acid cycle intermediates which can feed gluconeogenesis.

Ala, Cys, Gly, Ser,Asp, Asn, Glu, Gln. Thr depends upon the pathway.

AA Carbon Skeletons

Ketogenic amino acids degrade to acetylCoA or acetoacetylCoA which can contribute to the synthesis of fatty acids or ketone bodies.

Leu and Lys are the only two purely ketogenic amino acids.

Some amino acids yield both glucogenic and ketogenic parts.

Phe, Tyr, Trp, Ile. Thr depends upon the pathway.

Pink = glucogenic

Yellow = ketogenic

Pyruvate Family

-Ketoglutarate Family

All are converted to Glu first.

Histidine

Arginine and Proline

SuccinylCoA Family

All are converted to propionylCoA first and then follow the odd chain fatty acid pathway.

Thr with -ketoacid decarboxylase yields propionylCoA (an enzyme similar to PDH).

Methionine

Ile, Val and Leu

These non-polar, branched chain amino acids follow a similar sequence of three reactions:

1. Transamination giving an -ketoacid.

2. Oxidative decarboxylation giving an acylCoA.

3. AcylCoA dehydrogenase yields unsaturation.

The residue from Ile follows -ox to give acetylCoA and propionylCoA.

The residue from Val adds HOH, is then oxidized twice to yield an acid with a -carbonyl. This decarboxylates to give propionylCoA.

Leucine

Transaminase

-Keto acid decarboxylase

(Like PDH)

Leucine, cont.

AcylCoA dehydrogenase

Carboxylase

Leucine, cont.

Hydratase

Lyase

Conversion of Phe to Tyr

Requires tetrahydrobiopterin

Tetrahydrobiopterin

Formation and regeneration

Phe and Tyr Degradation

Tryptophan Degradation

Pool Molecules from Degradation

Amino Acid Pool Molecule

Asp, Asn Oxaloacetate

Ala, Cys, Gly, Ser, Thr, Trp Pyruvate

Arg, His, Pro, Gln, Glu -ketoglutarate

Met, Ile, Val, Thr SuccinylCoA

Asp, Phe, Tyr, Fumarate

Phe, Tyr, Trp, Ile, Leu, LysAcetylCoA and/orAcetoacetylCoA

End of Chapter 23

Copyright © 2007 by W. H. Freeman and Company

Berg • Tymoczko • Stryer

BiochemistrySixth Edition