Presentation on theme: "Protein Turnover and Amino Acid Catabolism"— Presentation transcript:
1 Protein Turnover and Amino Acid Catabolism Chem 454: Biochemistry IIUniversity of Wisconsin-Eau Claire
2 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 HereAmino acid metabolismUrea Cycle
3 Introduction Proteins are degraded into amino acids. Protein turnover is tightly regulated.First step in protein degradation is the removal of the nitrogenAmmonium ion is converted to urea in most mammals.Carbon atoms are converted to other major metabolic intermediates.Inborn errors in metabolism
4 IntroductionAmino acids used for synthesizing proteins are obtained by degrading other proteinsProteins 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?
5 Introduction Excess amino acids cannot be stored. Surplus amino acids are used for fuel.Carbon skeleton is converted toAcetyl–CoAAcetoacetyl–CoAPyruvateCitric acid cycle intermediateThe 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.
6 1. Protein DegradationDietary 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.
7 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.
8 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
9 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.
10 2.1 UbiquitinUbiquitin is activated and attached to proteins using a group of three enzymesE1 - Ubiquitin activating enzymeE2 - Ubiquitin-conjugating enyzmeE3 - 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.
11 3. Removal of NitrogenThe 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.
12 3.1 Conversion to Ammonium Ions α–Amino groups are converted to ammonium ions by the oxidative deamination of glutamate
13 3.1 TransaminationGenerally these enzyme funnel amino groups to α–ketoglutarate.Aspartate transaminaseAlanine transaminase- Aminotransferases (transaminases) catalyze the transfer of an α–amino acid to and α–keto acid.
14 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
15 3.1 DeaminationIn most terrestrial vertebrates the ammonium ion is converted to urea.
16 3.2 Pyridoxal PhosphatePyridoxal phosphate forms a Schiff-base intermediates in aminotransferase reactions.- We saw pyridoxal phosphate earlier in glycogen phosphorylase, where it played a different role.
17 3.2 Pyridoxyl PhosphatePyridoxyl phosphate can under go acid/base tautomerization.- The pyridine nitrogen is a weak base, while the phenolic OH is slightly acidic.
18 3.2 Pyridoxyl PhosphateThe 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
19 3.2 Pyridoxyl Phosphate Transamination mechanism: The second half of the reaction reverses these steps with a different α–keto acid.
20 3.2 Pyridoxyl PhosphatePyridoxyl phosphate is is a very versatile cofactorused to make bonds to Cα susceptible to cleavage.
21 3.4 Serine and ThreonineThe β–hydroxy amino acids, serine and threonine, can be directly deaminated
22 3.5 Transporting Nitrogen to Liver Urea is produced in the LiverThe alanine cycle is used to transport nitrogen to the liver- The alanine cycle is reminiscent of the Cori cycle.
23 4. Ammonium IonAmmonium ion is converted into urea in most terrestrial vertebrates
24 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 metabolismWe Are Here
25 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).
26 4.1 Formation of Carbamoyl Phosphate Carbamoyl synthetaseFree 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.
27 4.1 Formation of Citrulline Ornithine transcarbamoylaseCitrulline 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.
28 4.1 Formation of Arginosuccinate Condensation of citrulline with aspartate to form arginosuccinateTwo equivalent of ATP are required.- The α–amino group from the aspartate will become the second nitrogen for the urea
29 4.1 Formation of Arginine and Fumarate ArginosuccinaseCleaves arginosuccinate to form arginine and fumarate- This is an elimination reaction- Have produced an amino acid and an intermediate of the citric acid cycle.
30 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.
31 4.2 Linked to Citric Acid Cycle The urea cycle is linked to the citric acid cycle: Kreb’s Bi-cycle!!
32 5. Carbon AtomsThe carbon atoms of degraded amino acids emerge as major metabolic intermediates.Degradation of the 20 amino acids funnel into 7 metabolic intermediatesAcetyl–CoAAcetoacetyl–CoAPyruvateα-KetoglutarateSuccinyl–CoAFumarateOxaoloacetateKetogenicGlucogenic
48 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.(
49 6. Inborn Errors in Metabolism AlcaptonuriaAbsence of homogentisate oxidase activity- 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.(urinesclera
50 6. Inborn Errors in Metabolism TyrosinemiaAbsence of activity of fumarylacetoacetase
51 6. Inborn Errors in Metabolism AlbinismAbsence 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
52 6. Inborn Errors in Metabolism Maple syrup urine diseaseLack of branch-chain dehydrogenase activityLeads 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-dinitrophenylhydrazineAn isoleucine-, leucine- and valine-free unflavored powderdetection
53 6. Inborn Errors in Metabolism PhenylketonuriaAbsence 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