Molecular Biology: Protein Metabolism

Amino Acid Metabolism 2

• Ubiquitin is a small protein (76 amino acids) that targets proteins for destruction, present in all eukaryotic cells.
• Ubiquitin is highly conserved, attaching to the ε-amino groups of one or more lysine residues on the target protein via its carboxyl-terminal glycine residue.
• Ubiquitination requires ATP hydrolysis.
• The proteasome (26S proteasome) is a large, ATP-driven protease complex that digests ubiquitinated proteins.
• The 26S proteasome is a complex of a 20S catalytic unit arranged as a barrel and two 19S regulatory units that control access to the 20S catalytic subunit interior.
• The 19S regulatory units contain ubiquitin receptors that bind to polyubiquitin chains, use ATP to unfold polyubiquitinated chains and direct them into the catalytic core, and contain an isopeptidase that cleaves off intact ubiquitin molecules to be reused.

Proteolytic Active Sites of the 20S Barrel

• The proteasome's 20S barrel has three types of active sites in the β subunits, each with different specificity.
• All active sites employ an N-terminal threonine residue.
• The hydroxyl group of the threonine residue attacks the carbonyl groups of peptide bonds, forming acyl-enzyme intermediates.
• Substrates are degraded processively without releasing intermediate products.
• Substrates are processed into peptides ranging from seven to nine residues before release.

Proteasome and Other Proteases Generate Free Amino Acids

• Proteasomes, along with other proteases, break down proteins into their component amino acids, which can be used in other biosynthetic processes.
• The amino acids can be used for more synthesis or converted to other metabolic intermediates.

Processes Regulated by Protein Degradation

• Many biological processes are directly or indirectly controlled by the degradation of proteins.
• This control is crucial in various cellular and developmental processes.
  • Examples include gene transcription, cell-cycle progression, organ formation, circadian rhythms, inflammatory response, tumor suppression, cholesterol metabolism, and antigen processing.

Protein Degradation Can Be Used to Regulate Biological Function

• Bortezomib (Velcade) is a dipeptidyl boronic acid inhibitor of the proteasome, used as a therapy for multiple myeloma.
• HT1171 is an inhibitor of the proteasome of M. tuberculosis, having no effect on human proteasomes.

The First Step in Amino Acid Degradation Is the Removal of Nitrogen

• Amino acids not needed for building blocks are broken down into compounds for the metabolic cycle.
• The amino group is removed, and the remaining carbon skeleton is converted to a glycolytic intermediate or acetyl CoA.
• The liver is the primary site of amino acid degradation in mammals.
• Muscles also readily degrade branched-chain amino acids (leucine, isoleucine, and valine).

Fate of Amino Acids in Liver and Cells

• Dietary proteins are broken down to amino acids in the gut.
• Amino acids are used in protein synthesis, glucose synthesis, or conversion to fatty acids .
• Branched-chain amino acids are primarily oxidized in skeletal muscle.

Alpha-Amino Groups of Many Amino Acids Are Converted into Ammonium Ions by the Oxidative Deamination of Glutamate in the Liver

• The alpha-amino group is transferred to a-ketoglutarate, producing glutamate.
• Glutamate is oxidatively deaminated in the liver to produce ammonium ions (NH4+).

The Role of Aminotransferases

• Aminotransferases (transaminases) catalyze the reversible transfer of an alpha-amino group from one amino acid to an alpha-keto acid.
• This reaction is reversible, so it can be utilized to generate amino acids from alpha-keto acids.

The Role of Glutamate Dehydrogenase

• Glutamate dehydrogenase is a mitochondrial enzyme that converts the nitrogen atom in glutamate to a free ammonium ion (NH4+) via oxidative deamination.
• It can use either NAD+ or NADP+ and is a liver-specific enzyme.
• It is allosterically inhibited by GTP and stimulated by ADP.

The Fate of the Ammonia Ion

• The sum of the reactions of aminotransferases and glutamate dehydrogenase produces NH4+ (ammonia) from alpha-amino acids.
• In most terrestrial vertebrates, ammonia (NH4+) is converted into urea, which is then excreted.

Serine and Threonine Can Be Directly Deaminated

• Serine dehydratase and threonine dehydratase directly deaminate serine and threonine to pyruvates and alpha-ketobutyrate, respectively—both of these reactions have PLP (pyridoxal phosphate) as a prosthetic group.

Protein turnover—the degradation and resynthesis of proteins

• Protein turnover refers to the degradation and resynthesis processes of proteins within an organism continuously.

Protein Turnover—an Index of Basal Metabolism

• Protein turnover is a vital aspect of basal metabolism.

Regulation of Protein Turnover

• Various factors, including hormones, nutrients, and physical activity, regulate protein turnover.
• Multiple tissues and organs contribute to protein synthesis and degradation in the body.

Peripheral Tissues Transport Nitrogen to the Liver

• Muscle uses branched-chain amino acids as fuel during prolonged exercise and fasting.
• Muscle lacks urea cycle enzymes.
• Nitrogen is transported from muscle to the liver as alanine via the glucose-alanine cycle.
• Amino groups from amino acids can also be transferred to glutamine, then processed in the liver.

Pathway Integration: The Glucose-Alanine Cycle

• This cycle allows muscle to use amino acids as fuel during prolonged exercise and fasting.
• Muscle releases nitrogen in the form of alanine, and the alanine is utilized in the liver to produce glucose.

How Alanine Is Used to Transfer Amino Groups to the Liver

• Alanine is essential for transferring amino groups from muscles to the liver.

Ammonium Ions Are Converted into Urea in Most Terrestrial Vertebrates

• Some of the ammonia produced in amino acid breakdown is consumed in the biosynthesis of nitrogen compounds.
• Most terrestrial vertebrates convert excess ammonia into urea via the urea cycle for excretion (ammonia, on the other hand, is toxic, so conversion is a necessity).

Glutamate Dehydrogenase: A Major Source of NH4+ in Liver

• Glutamate dehydrogenase plays a critical role in urea production by releasing ammonia.

Where Does the NH4+ Go?

• Ammonia (NH4+) is a byproduct of amino acid break down.

The Urea Cycle Eliminates Both Nitrogen and Carbon Waste Products

• The urea cycle breaks down carbon and nitrogen waste and eliminates it through the formation of urea.

The Urea Cycle: Straddling the Mitochondria and the Cytoplasm

• The urea cycle is a central metabolic process that occurs both in the cytoplasm and the mitochondria.
• The cycle involves numerous enzymes that synthesize urea by incorporating the nitrogen source from various metabolic processes.
• The cycle regenerates substrates used in intermediary metabolism.

The Urea Cycle Begins with the Formation of Carbamoyl Phosphate

• Carbamoyl phosphate synthetase I catalyzes the coupling of ammonia and bicarbonate to form carbamoyl phosphate.
• This reaction requires ATP and takes place in the mitochondria.

Carbamoyl Phosphate Synthetase I Is the Key Regulatory Enzyme for Urea Synthesis

• Carbamoyl phosphate synthetase I requires N-acetylglutamate (NAG) for activity.
• NAG synthesis is stimulated when arginine levels are high.
• CPSI is allosterically regulated by NAG and can be regulated by covalent modification.

The Sirtuin Family of Protein Deacetylases—Role of SIRT 3, 4, and 5 in the Urea cycle

• Sirtuins play a role in amino acid degradation and urea cycle regulation.
• Specific sirtuins affect amino acid degradation and urea cycle activity by deacetylating specific targets.
• Sirtuin activity is dependent on NAD+ levels in the cell.

Control of Flux through the Urea Cycle

• Several factors control the rate of urea cycle flux.
• High protein intake and glucocorticoids increase urea cycle enzyme expression and activity
• Other factors, like the ratios of NAD+/NADH and ADP/ATP, also affect the regulatory steps.

Inherited Defects of the Urea Cycle

• Humans excrete a considerable amount of urea.
• Defects in the urea cycle cause elevated NH₄⁺ blood levels (hyperammonemia), which is toxic.
• Symptoms of urea cycle defects may include lethargy, vomiting, coma, and potentially irreversible brain damage (hepatic encephalopathy).
• Elevated NH₄⁺ can disrupt neurotransmitter systems, energy metabolism, and cellular osmolality, leading to various consequences.

Argininosuccinase Deficiency Can Be Managed by Supplementing the Diet with Arginine.

• Argininosuccinase deficiency can be managed by supplementation with arginine and restriction of protein intake to prevent excess nitrogen build-up.

Urea Is Not the Only Means of Disposing of Excess Nitrogen

• Ammoniotelic organisms excrete ammonia.
• Uricotelic organisms excrete uric acid.

Summary

• The proteasome breaks down proteins that are no longer needed or have been damaged.
• Amino acids in excess of body needs or immediate requirements are deaminated, to produce urea for excretion.
• The carbon skeletons of amino acids can be used or regenerated.
• The glucose-alanine cycle allows muscles to transport nitrogen to the liver and participate in glucose synthesis.
• Urea synthesis is catalyzed by enzymes in the urea cycle and ultimately produces urea for excretion from the body.