Protein Turnover and Amino Acid Catabolism. The Digestion and Absorption of Dietary Proteins Pepsin...

Protein Turnover and Amino Acid Catabolism

The Digestion and Absorption of Dietary Proteins

• Pepsin nonspecific maximally active at low pH of the stomach.

• Proteolytic enzymes of the pancreas in the intestinal lumen display a wide array of specificity.

Aminopeptidases digest proteins from the amino-terminal end.

Cellular Proteins Are Degraded at Different Rates

•Some proteins are very stable, while others are short lived.

–Altering the amounts of proteins that are important in metabolic regulation can rapidly change

metabolic patterns.

•Cells have mechanisms for detecting and removing damaged proteins.

–A significant proportion of newly synthesized protein molecules are defective because of errors in

translation.–Other proteins may undergo oxidative damage or

be altered in other ways with the passage of time.

Ubiquitin Tags Proteins for Destruction

•How can a cell distinguish proteins that are meant for

degradation?•Ubiquitin, a small (8.5-kd) protein

present in all eukaryotic cells, is the tag that marks proteins for

destruction.

•The c-terminal glycine residue of ubiquitin (Ub) becomes covalently attached to the -amino groups of

several lysine residues on a protein destined to be degraded.

•The energy for the formation of these isopeptide bonds (iso

because- rather than -amino groups are targeted) comes from

ATP hydrolysis.

•Three enzymes participate in the attachment of ubiquitin to each protein:

–ubiquitin-activating enzyme, or E1–ubiquitin-conjugating enzyme, or E2–ubiquitin-protein ligase, or E3 .

•Chains of ubiquitin can be generated by the

linkage of the -amino group of lysine residue

48 of one ubiquitin molecule to the terminal

carboxylate of another.•Chains of four or more Chains of four or more

ubiquitin molecules are ubiquitin molecules are particularly effective in particularly effective in signaling degradationsignaling degradation

What determines whether a protein becomes ubiquitinated?

.1The half-life of a cytosolic protein is determined to a large extent by its amino-terminal residue “the N-terminal rule.”

–A yeast protein with methionine at its N terminus typically has a half-life of more than 20 hours, whereas one with arginine at

this position has a half-life of about 2 minutes.•A highly destabilizing N-terminal residue such as arginine

or leucine favors rapid ubiquitination, whereas a stabilizing residue such as methionine or proline does not.

•E3 enzymes are the readers of N-terminal residues..2Cyclin destruction boxes are amino acid sequences that

mark cell-cycle proteins for destruction..3Proteins rich in proline, glutamic acid, serine, and

threonine (PEST sequences).

The Proteasome Digests the Ubiquitin-Tagged Proteins

•A large protease complex called the proteasome or the 26S proteasome digests the ubiquitinated

proteins.•This ATP-driven multisubunit protease spares

ubiquitin, which is then recycled.•The 26S proteasome is a complex of two

components:–20S proteasome, which contains the catalytic

activity1–19S regulatory subunit .

• The OH groups of these aas are converted into nucleophiles with the assistance of their own amino groups.

• These nucleophilic groups then attack the carbonyl groups of peptide bonds and form acyl-enzyme intermediates.

• ATP hydrolysis may assist the 19S complex to unfold the substrate and induce conformational changes in the 20S proteasome so that the substrate can be passed into the center of the complex

Protein Degradation Can Be Used to Regulate Biological

Function

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Digested proteins

Amino Acids

Degradation in the liver

NH4+ -ketoacids

enter the metabolic mainstream as precursors

to glucose or citric acid cycle intermediates

The amino group must be removed, as there are no nitrogenous compounds in energy-transduction pathways

The fate of the -amino group

•The -amino group of many aas is transferred to -ketoglutarate to form glutamate.

•Glutamate is then oxidatively deaminated to yield ammonium ion (NH4

+).

•Aminotransferases (transaminases) catalyze the transfer of an -amino group from an

-amino acid to an -keto acid.

Example:

•Aspartate aminotransferase:

•Alanine aminotransferase:

•These transamination reactions are reversible and can thus be used to synthesize amino acids

from a-ketoacids ,

•The nitrogen atom that is transferred to -ketoglutarate in the transamination reaction is

converted into free ammonium ion by oxidative deamination.

•This reaction is catalyzed by glutamate dehydrogenase.

•This enzyme is unusual in being able to utilize either NAD+ or NADP+ at least in some species.

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

resulting Schiff base.

•Glutamate dehydrogenase and other enzymes required for the production of urea are located in

mitochondria.•This compartmentalization sequesters free

ammonia, which is toxic.

•In most terrestrial vertebrates, NH4+ is converted

into urea, which is excreted .

Pyridoxal Phosphate Forms Schiff-Base Intermediates in

Aminotransferases•All aminotransferases contain

the prosthetic group pyridoxal phosphate (PLP), which is

derived from pyridoxine (vitamin B6).

Pyridoxal phosphate derivatives can form stable tautomeric forms

a pyridine ring that is slightly basic

A phenolic hydroxyl group that is slightly acidic

The most important functional group allows PLP to form covalent Schiff-base intermediates with amino acid substrates

•The aldehyde group of PLP usually forms a Schiff-base linkage with the -amino group of a

specific lysine residue of the enzyme.

•The -amino group of the amino acid substrate displaces the -amino group of the active-site

lysine residue .

-ketoglutarate

•Some of the NH4+ formed in the breakdown of

amino acids is consumed in the biosynthesis of nitrogen compounds.

•In most terrestrial vertebrates, the excess NH4+ is

converted into urea and then excreted.

•The urea:–One nitrogen atom is transferred from aspartate.–The other nitrogen atom is derived directly from free

NH4 +.

–The carbon atom comes from HCO3-.

The Urea Cycle

The Urea Cycle Reactions

.1Formation of Carbamoyl Phosphate: catalyzed by carbamoyl phosphate synthetase.

•The consumption of two molecules of ATP makes the synthesis essentially irreversible.

•The carbamoyl group of carbamoyl phosphate has a high transfer potential because of its

anhydride bond.

.2Carbamoyl is transferred to ornithine to form citrulline.

•The reaction is catalyzed by ornithine transcarbamoylase.

•Ornithine and citrulline are amino acids, but they are not used as building blocks of

proteins.

.3Citrulline is transported to the cytoplasm where it condenses with aspartate to form

argininosuccinate•The reaction is catalyzed by argininosuccinate

synthetase.•The reaction is driven by the cleavage of ATP

into AMP and PPi, and by the subsequent hydrolysis of PPi.

.4Argininosuccinase cleaves argininosuccinate into arginine and fumarate.

•Thus, the carbon skeleton of aspartate is preserved in the form of fumarate.

.5Arginine is hydrolyzed to generate urea and ornithine in a reaction catalyzed by arginase.

•Ornithine is then transported back into the mitochondrion to begin another cycle.

•Mitochondrial reactions:–The formation of NH4+ by glutamate

dehydrogenase.–Its incorporation into carbamoyl phosphate–Synthesis of citrulline

•Cytosolic reactions:–The next three reactions of the urea cycle,

which lead to the formation of urea, take place in the cytosol .