Protein Turnover and Amino Acid Catabolism Chem 454: Biochemistry II University of Wisconsin-Eau Claire

We Are Here Urea Cycle Amino acid metabolism - Proposed by Hans Krebs and Kurt Henseleit in 1932 - Was the first metabolic cycle to be discovered. • Before the citric acid cycle • It is linked to the citric acid cycle. - One of the N atoms comes directly from ammonium ion, the other from aspartate. - The C atom comes from HCO3 (CO2). We Are Here Amino acid metabolism Urea Cycle

Introduction Proteins are degraded into amino acids. Protein turnover is tightly regulated. First step in protein degradation is the removal of the nitrogen Ammonium ion is converted to urea in most mammals. Carbon atoms are converted to other major metabolic intermediates. Inborn errors in metabolism

Introduction Amino acids used for synthesizing proteins are obtained by degrading other proteins Proteins destined for degradation are labeled with ubiquitin. Polyubiquinated proteins are degraded by proteosomes. Amino acids are also a source of nitrogen for other biomolecules. - What is an example of another type of biomolecule that requires nitrogen?

Introduction Excess amino acids cannot be stored. Surplus amino acids are used for fuel. Carbon skeleton is converted to Acetyl–CoA Acetoacetyl–CoA Pyruvate Citric acid cycle intermediate The amino group nitrogen is converted to urea and excreted. Glucose, fatty acids and ketone bodies can be formed from amino acids. - Unlike glucose and fatty acids, which can be stored.

1. Protein Degradation Dietary proteins are a vital source of amino acids. Discarded cellular proteins are another source of amino acids. - Proteins a re hydrolyzed in the stomach and small intestines and the component amino acids absorbed into the bloodstream.

1.1 Dietary Protein Degradation Dietary proteins are hydrolyzed to amino acids and absorbed into the bloodstream. - Protein digestion starts in the stomach - acid hydrolysis - Proteases are synthesized as zymogen and then activated in the stomach and small instestine. - Aminopeptidase N is located in the plasma membrane of the instestinal cells.

1.2 Cellular Protein Degradation Cellular proteins are degraded at different rates. Ornithine decarboxylase has a half-life of 11 minutes. Hemoglobin lasts as long as a red blood cell. Υ-Crystallin (eye lens protein) lasts as long as the organism does. - Ornithine decarboxylase has a half-life of 11 minutes - Gamma-crystalline lasts the lifetime of the organism

2. Regulation of Protein Turnover The protein ubiquitin is used to mark cellular proteins for destruction. - The ubiquitin is attached to the ε-amino groups of lysines by an isopeptide bond to the C-terminus of ubiquitin. - Ubiquitin is highly conserved throughout evolution: yeast and humans differ by only 3 out of 76 amino acids.

2.1 Ubiquitin Ubiquitin is activated and attached to proteins using a group of three enzymes E1 - Ubiquitin activating enzyme E2 - Ubiquitin-conjugating enyzme E3 - Ubiquitin-protein ligase - Reactions are similar to those that activate acyl groups in fatty acid degradation. - There are only one or a small number of distinct E1 proteins. - Eukaryotes have many distinct E2 and E3 proteins. - Only a single family of E2 proteins, but many families of E3 proteins. • E3 provides most of the substrate specificity. Combination of E2 and E3 allow for fine tuning of specificity. - The human papilloma virus encodes for an E3 protein which targets the p53 tumor suppressor protein in its host. 90% of the cervical cancers are associtated with this type of activity. The human papilloma virus encodes for an E3 protein which targets the p53 tumor suppressor protein in its host. 90% of the cervical cancers are associtated with this type of activity.

3. Removal of Nitrogen The first step in amino acid degradation is the removal of the nitrogen. The liver is the major site of protein degradation in mammals. Deamination produces α-keto acids, which are degraded to other metabolic intermediates.

3.1 Conversion to Ammonium Ions α–Amino groups are converted to ammonium ions by the oxidative deamination of glutamate

3.1 Transamination Generally these enzyme funnel amino groups to α–ketoglutarate. Aspartate transaminase Alanine transaminase - Aminotransferases (transaminases) catalyze the transfer of an α–amino acid to and α–keto acid.

3.1 Deamination Glutamate dehydrogenase - Uses either NAD or NADP - Takes place in the mitochondria, where the toxic effects of the ammonium ion can be isolated - Removal of the ammonium ion pulls this reaction to the right. - Lowering the energy charge accelerates the oxidation of amino acids • - GTP, ATP • + GDP, ADP

3.1 Deamination In most terrestrial vertebrates the ammonium ion is converted to urea.

3.2 Pyridoxal Phosphate Pyridoxal phosphate forms a Schiff-base intermediates in aminotransferase reactions. - We saw pyridoxal phosphate earlier in glycogen phosphorylase, where it played a different role.

3.2 Pyridoxyl Phosphate Pyridoxyl phosphate can under go acid/base tautomerization. - The pyridine nitrogen is a weak base, while the phenolic OH is slightly acidic.

3.2 Pyridoxyl Phosphate The aldehyde forms a Schiff–base with an ε–amino group on the enzyme. This Schiff-bases can be exchanged for one with the α–amino group of an amino acid - The Schiff-base becomes protonated and the charge is stabilized by the phenolate ion. - The Schiff-base is also called an aldimine

3.2 Pyridoxyl Phosphate Transamination mechanism: The second half of the reaction reverses these steps with a different α–keto acid.

3.2 Pyridoxyl Phosphate Pyridoxyl phosphate is is a very versatile cofactor used to make bonds to Cα susceptible to cleavage.

3.4 Serine and Threonine The β–hydroxy amino acids, serine and threonine, can be directly deaminated

3.5 Transporting Nitrogen to Liver Urea is produced in the Liver The alanine cycle is used to transport nitrogen to the liver - The alanine cycle is reminiscent of the Cori cycle.

4. Ammonium Ion Ammonium ion is converted into urea in most terrestrial vertebrates

4. The Urea Cycle:reminder - Proposed by Hans Krebs and Kurt Henseleit in 1932 - Was the first metabolic cycle to be discovered. • Before the citric acid cycle • It is linked to the citric acid cycle. - One of the N atoms comes directly from ammonium ion, the other from aspartate. - The C atom comes from HCO3 (CO2). Amino acid metabolism We Are Here

4. The Urea Cycle - Proposed by Hans Krebs and Kurt Henseleit in 1932 - Was the first metabolic cycle to be discovered. • Before the citric acid cycle • It is linked to the citric acid cycle. - One of the N atoms comes directly from ammonium ion, the other from aspartate. - The C atom comes from HCO3 (CO2).

4.1 Formation of Carbamoyl Phosphate Carbamoyl synthetase Free NH4 reacts with HCO3 to form carbamoyl phosophate. Reaction is driven by the hydrolysis of two molecules of ATP - The mammalian enzyme requires the allosteric effector, N-Acetylglutamate for activity.

4.1 Formation of Citrulline Ornithine transcarbamoylase Citrulline is formed from transfer of the carbamoyl group to the γ-amino group of ornithine. - Both ornithine and citrulline are α–amino acids. - The formation of NH4+ from glutamate dehydrogenase and the subsequent incorporation into the citrulline occurs in the mitochondrial matrix -The other reactions of the urea cycle take place in the cytosol -Citrulline is transported to the cytosol.

4.1 Formation of Arginosuccinate Condensation of citrulline with aspartate to form arginosuccinate Two equivalent of ATP are required. - The α–amino group from the aspartate will become the second nitrogen for the urea

4.1 Formation of Arginine and Fumarate Arginosuccinase Cleaves arginosuccinate to form arginine and fumarate - This is an elimination reaction - Have produced an amino acid and an intermediate of the citric acid cycle.

4.1 Formation of Urea Arginase The arginine is hydrolyzed to produce the urea and to reform the ornithine. The ornithine reenters the mitochondrial matrix.

4.2 Linked to Citric Acid Cycle The urea cycle is linked to the citric acid cycle: Kreb’s Bi-cycle!!

5. Carbon Atoms The carbon atoms of degraded amino acids emerge as major metabolic intermediates. Degradation of the 20 amino acids funnel into 7 metabolic intermediates Acetyl–CoA Acetoacetyl–CoA Pyruvate α-Ketoglutarate Succinyl–CoA Fumarate Oxaoloacetate Ketogenic Glucogenic

5. Carbon Atoms Ketogenic Glucogenic Both leucine lysine serine threonine aspartic acid glutamic acid asparagine glutamine glycine alanine valine proline histidine arginine methionine cysteine Both isoleucine phenylalanine tryptophan tyrosine

Class problem Explain the meaning (from a biochemistry perspective) of the saying “fats burn in the flame of carbohydrates.” How would proteins fit into this statement?

5. Carbon Atoms - Alanine -

5.1 Pyruvate Entry Point - Know • Alanine - transmination • Serine - deamination

5.2 Oxaloacetate Entry Point Aspartate Transamination to oxaloacetate Asparagine Hydrolysis to Aspartate + NH4+ Transmination to oxaloacetate

5.3 α–Ketoglutarate Entry Point Five carbon amino acids

5.3 α–Ketoglutarate Entry Point Histidine

5.3 α–Ketoglutarate Entry Point Proline and Arginine

5.4 Succinyl–CoA Entry Point Methionine, Valine & Isoleucine

5.4 Succinyl–CoA Entry Point Methionine Forms S-Adenosylmethionine S-Adenosylmethionine is

5.6 Branched-chained Amino Acids - The oxidative decarboxylation occurs using a commplex that is similar to pyruvate and alpha-ketoglutarate dehydrogenases.

5.7 Aromatic Amino Acids Phenylalanine

5.7 Aromatic Amino Acids Tetrahydrobiopterin - electron carrier

5.7 Aromatic Amino Acids Phenylalanine & Tyrosine - p-hydroxyphenylpyruvate reacts with a dioxygenase to produce homogentisate - the homogentisate reacts with an dioxygenase to cleave it.

5.7 Aromatic Amino Acids Tryptophan

6. Inborn Errors in Metabolism Tyrosine related disorders - Discovered in 1902 by Archibald Gerrod as a mendelian inherited dissease - Urine turns dark onstanding - Show student the Online Mendelian Inheritance in Man (OMIM) database.(http://www.ncbi.nlm.nih.gov/Omim/)

6. Inborn Errors in Metabolism Alcaptonuria Absence of homogentisate oxidase activity http://www.emedicine.com/ped/topic64.htm - Discovered in 1902 by Archibald Gerrod as a mendelian inherited dissease - Urine turns dark onstanding - Show student the Online Mendelian Inheritance in Man (OMIM) database.(http://www.ncbi.nlm.nih.gov/Omim/) urine sclera

6. Inborn Errors in Metabolism Tyrosinemia Absence of activity of fumarylacetoacetase http://www.childrenshospital.org/newenglandconsortium/NBS/descriptions/tyro1.html http://www.myspecialdiet.com/Shop/Search.aspx?t=department&i=14

6. Inborn Errors in Metabolism Albinism Absence of melanin pigment - Phenylalanine is not converted to tyrosine - Forms phenylpyruvate - Initially described in 1934 - Observed by reacting phenylpyruvate with FeCl(3) (turns olive green) - Leads to sever retardation http://home.clara.net/knowlton/family/Albinism/bianca.htm http://www.nlm.nih.gov/medlineplus/ency/article/001166.htm

6. Inborn Errors in Metabolism http://www.nlm.nih.gov/medlineplus/ency/article/000373.htm Maple syrup urine disease Lack of branch-chain dehydrogenase activity Leads to elevation of α–keto banched-chain acids (branched-chain keto aciduria) - Urine smells like maple syrup - Mental retardatioin unless put on a diet early in life that is low in valine, isoleucine and leucine - Detect with 2,4-dinitrophenylhydrazine An isoleucine-, leucine- and valine-free unflavored powder detection

6. Inborn Errors in Metabolism Phenylketonuria Absence of phenylalanine hydroxylase activity - Phenylalanine is not converted to tyrosine - Forms phenylpyruvate - Initially described in 1934 - Observed by reacting phenylpyruvate with FeCl(3) (turns olive green) - Leads to severe retardation http://www.nlm.nih.gov/medlineplus/ency/article/001166.htm

6. Inborn Errors in Metabolism