Protein Metabolism Denotes the various biochemical processes responsible for the synthesis of proteins and amino acids the breakdown of proteins (and other large molecules, too) by 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 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 e-amino groups of several lysine residues on a protein destined to be degraded. The energy for the formation of these isopeptide bonds (iso because e- rather than a-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 e-amino group of lysine residue 48 of one ubiquitin molecule to the terminal carboxylate of another. Chains of four or more ubiquitin molecules are particularly effective in signaling degradation What determines whether a protein becomes ubiquitinated?
The half-life of a cytosolic protein is determined to a large extent by its amino-terminal residue the N-terminal rule. In yeast: if N terminus is methionine half-life > 20 hours, whereas if N terminus is arginine half-life 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. Cyclin destruction boxes are amino acid sequences that mark cell-cycle proteins for destruction. Proteins 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. In eukaryotes, they are located in the nucleus and the cytoplasm. The degradation process yields peptides of about 7-8 amino acids long, then further degraded into amino acids and used in synthesizing new proteins. This ATP-driven multisubunit protease spares ubiquitin, which is then recycled. Protein Degradation Can Be Used to Regulate Biological Function
Example: E3 P P E3 NF-kB I-kB P Inflammation Ub initiates the expression of a number of the genes that take part in this response P Ub NF-kB proteosome What is the fate of amino acids released on protein digestion?
Nitrogen Removal is the first step in the degradation of amino acids. Any amino acids not needed as building blocks are degraded to various compounds, depending on the type of amino acid and the tissue from which it originates. The major site of amino acid degradation in mammals is the liver. The resulting -ketoacids are then metabolized so that the carbon skeletons can: 1. enter the metabolic mainstream as precursors of glucose 2. or as citric acid cycle intermediates. Degradation in the liver
Digested proteins Amino Acids Degradation in the liver NH4+ a-ketoacids The amino group must be removed, as there are no nitrogenous compounds in energy-transduction pathways enter the metabolic mainstream as precursors to glucose or citric acid cycle intermediates The fate of the a-amino group
The a-amino group of many aas is transferred to a-ketoglutarate to form glutamate. Glutamate is then oxidatively deaminated to yield ammonium ion (NH4+). Aminotransferases (transaminases) catalyze the transfer of an a-amino group from an a-amino acid to an a-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 a-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. Exception the -amino groups of serine and threonine can be directly converted into NH4+ . These direct deaminations are catalyzed by serine dehydratase and threonine dehydratase, in which pyridoxal phosphate (PLP) is the prosthetic group. 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
The most important functional group allows PLP to form covalent Schiff-base intermediates with amino acid substrates a pyridine ring that is slightly basic A phenolic hydroxyl group that is slightly acidic The aldehyde group of PLP usually forms a Schiff-base linkage with the e-amino group of a specific lysine residue of the enzyme. The a-amino group of the amino acid substrate displaces the e-amino group of the active-site lysine residue. Peripheral Tissues Transport Nitrogen to the Liver
Muscle uses branched-chain amino acids: - leucine, - Valine, - Isoleucine as a source of fuel during prolonged exercise and fasting. How is the nitrogen processed in these other tissues?
As in the liver - the first step is removal of nitrogen from the amino acid. - However, muscle lacks the enzymes of the urea cycle (the set of reactions that prepares nitrogen for excretion). in muscle, the nitrogen must be released in a form that can be absorbed by the liver and converted into urea. Nitrogen is transported from muscle to the liver in two principal transport forms:
Alanine and glutamine. Glutamate is formed by transamination reactions, - Nitrogen is then transferred to pyruvate to form alanine, which is released into the blood. The liver takes up the alanine and converts it back into pyruvate by transamination.
The pyruvate can be used for gluconeogenesis, and the amino group eventually appears as urea. This transport is referred to as the glucosealanine cycle. It is reminiscent of the Cori cycle
It is reminiscent of the Cori cycle. However, in contrast with the Cori cycle, pyruvate is not reduced to lactate, Thus more high-energy electrons are available for oxidative phosphorylation in muscle. Glutamine synthase catalyzes the synthesis of glutamine from glutamate and NH4+ in an
ATP-dependent reaction: The nitrogen atoms of glutamine can be converted into urea in the liver The Urea Cycle 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 Reactions
Formation 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. Carbamoyl 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. Citrulline 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. Argininosuccinase cleaves argininosuccinate into arginine and fumarate.
Thus, the carbon skeleton of aspartate is preserved in the form of fumarate. Arginine 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. THE END