Metabolism of Proteins & Amino Acid Nitrogen 7 Amino acids - structure, properties. Intracellular degradation of proteins - proteasome, ubiquitin, lysosome. Transamination. Deamination. Ureosynthesis. Metabolism of Proteins - Overview Amines Neurotransmitters Purines/pyrimidines Porphyrins Intermediates of CAC A Glucose Fatty acids Lipids C02, H20 Energy Glycogen * proteins with various half-life time (minutes - several days) Intracellular Degradation of Proteins a) Lysosomal Protein Turnover ■ Proteins degraded: extracellular (accepted by endocytosis), membrane bonded, intracellular under the stress (autophagy) b) Ubiquitin - Proteasome Pathway (cytoplasm, nucleus) ■ Proteins degraded: damaged or misfolded intracellular proteins proteins coded by viruses and other intracellular parasites transcription factors cyclins and other regulation proteins proteins with the short half-life Proteasome ■ Important for cell processes (growth, differentiation, signal transduction, apoptosis). 1. Describe the steps in extracellular glycoproteins degradation. 2. What is the consequence of plasmatic glycoproteins desialization? 3. What is the function of ubiquitin in cells? 4. Describe the structure and function of proteasome. 37 Activation of target protein by ubiquitin NH, + Protein targeted for degradation Activated ubiquitin-protein ligase o Activated ubiquitin HS-(J) (complete) Target protein-ubiquitin complex bonded by amide bond Regulation of protein degradation - levels: activation of target protein by ubiquitin activation of ubiquitin-protein ligase 5. It was described more then 300 of different ubiquitin ligases in the cell. Each of them has targets a different kind structurally aberrant protein, protein with the short half-life time or regulatory proteins. What will be the consequence of missing of certain ubiquitin ligase in the cell? 6. Cell cycle is coordinated by cyclin-dependent kinases. Most of cyclins has half-life time about 0.5-1 hour. Which of degradation processes are involved in degradation of cyclins and what significance does it have. 7. There is known only one proteasome inhibitor used in therapy. It is synthetic tripeptide containing boron (bortezomib). It is approved in the U.S. for treating relapsed multiple myeloma. Try to explain the principle of its effect on the myeloma cells. 8. Which of protein degradation processes is ATP dependent? Elimination of a-Amino Nitrogen from Amino Acids 1) Transamination - most of amino acids except: Arg, Lys, Met, Thr, Tip, Pro, His ■ General equation of transamination reaction (complete): COOH H2N—CH I COOH I o=c 9. Which of the 2-oxo acids is most common acceptor of amino group? 10. What cofactor is used by amino transferases? 38 ] 1. Write the equation of a reaction catalyzed by alanine aminotransferase (ALT). 12. Write the equation of a reaction at which is formed Asp in a reaction catalyzed by aspartate aminotransferase (AST). 13. What is the fate of amino acid that is formed in both the reaction catalyzed by ALT, AST? 14. Explain the general significance of aminotransferases in amino acid degradation. 15. Complete the steps in transamination reaction: 2) Deamination - only some amino acids a) Deamination Associated with Dehydrogenation ■ Glutamate dehydrogenase (GMD) NAD(P)+ NAD(P)H+ HP ^° L-Glutamate 2-Iminoglutarate 2-Oxoglutarate Reaction is reversible, NAD+ is utilized mainly at deamination reaction, while NADP+ at the synthesis. 16. At which conditions will the reaction occur in opposite direction (formation of L-glutamate)? 17. Formation of glutamate in glutamate dehydrogenase catalyzed reaction is associated with decrease of energy production, especially in brain. Explain it. 18. Where in the cell is GMD found? 39 ■ L-Aminooxidases (FMN); D-aminooxidases (FAD, kidney, liver) Flavine Flavine H2 ^° ^3 R—CH—COOH « ............. "*- 2-Imino acid 2-Oxoacid 19. What significance D-aminooxidases have for human organism? b) Histidine Deamination Associated with Desaturation 20. The product of histidine deamination is urocanic acid (double bond between a- and p-carbon). Draw the structure. Detoxification of Ammonia ■ Plasmatic concentration of (NH4+and NH3): usually 6-12 umol/L (limit < 50 umol/L) a) Ureosynthesis (liver) 21. Ammonia is transported from muscle into the liver mainly in form of alanine a glutamine. Describe reactions at which is ammonia in liver released from these amino acids. 22. Write the equation summarizing the formation of 1 mol of urea. 23. What is the origin of two nitrogen atoms in the structure of urea? 24. What is the ATP consumption at the synthesis of 1 mol of urea? 25. What parts of a cell are processes of urea synthesis located in? 26. Partial reaction of ureosynthetic cycle (complete): • Carbamoyl phosphate synthesis NH3 + HCO3 +...... -*- ..............+..... +..... Carbamoyl phosphate • Citrulline formation H2C-NHo I CH2 CH2 I Carbamoyl phosphate + HC—NH2 C00H Citrulline 40 • Argininosuccinate formation ATP AMP + PPj Citrulline + Argininosuccinate • Cleavage of argininosuccinate Argininosuccinate + Fumarate • Arginine cleavage L-Arginine H20 L-Ornithine 27. By which reaction can fumarate convert to L-aspartate? 28. Draw the structure of a compound that serves as allosteric activator of carbamoyl phosphate synthase I. 29. L-aspartate is consumed during the urea synthesis. How can it resynthetized from fumarate that is formed during urea synthesis? 30. Urea synthesis is acidifying process. Explain, why. 31. Urea is bonded to Abends of proteins in non-enzymic reaction (carbamylation of proteins). Draw the structure of products that are formed by carbamylation of haemoglobin amino end Hb-Val-NH^ (limit up to 1.6 % of the total Hb(4Fe)). b) Glutamine Synthetase (muscle, brain, liver, mitochondria) 32. Detoxification of ammonia in brain occurs mainly by its binding to glutamate. What reaction supplies the consumed glutamate? c) Glutamate Dehydrogenase 33. Give the name of substrate that binds ammonia in tissues in reaction catalyzed by GMD. Does this reaction require energy? Is it oxidation or reduction? ATP ADP + Pi L-Glutamate L-Glutamine NH,+ 41 Metabolism of Amino Acids 8 Structure and names of amino acids, main pathways of their metabolism. Cofactors of transamination, decarboxylation, transfer one-carbon units, oxidation, reduction. Overview of Catabolism of Amino Acid Carbon Skeletons Thr glukosa A PEP Ala <- Al Trp Cys, Gly, Ser Pyruvate He co2 * Thr Acetyl-CoA Asn, Asp < > Oxalacetate Phe, Tyr —> Fumarate r Lys Acetoacetyl-CoA - > Acetoacetate HMG-CoA ~~Ť Leu Tyr, Phe 2-Oxoglutarate Glu < Succinyl-CoA i Propionyl-CoA His, Pro, Orn, Arg Val, Met, He, (Thr) 1. Which of amino acids are only ketogenic, keto- and glucogenic, only glucogenic? 2. Which of amino acids give acetyl-CoA by their metabolism? 3. Which of amino acids give propionyl-CoA by their metabolism? In which way is this compound metabolized? What cofactor is necessary? 4. What is the cofactors of: a) aminotransferases; b) decarboxylases. 5. Give the examples of decarboxylases of amino acids and their products. Complete the missing formulas and names in the following diagrams: 42 Alanine Ala ALT Arginine A. Urea cycle Arg H,0 + B. NO formation Arg oxidi"ion , NO NO-synthase Citrulline C. Creatine phosphate formation Arg Guanidoacetate ATP ADP Creatine phosphate 6. What amino acids are necessary for creatine synthesis? 7. Arg does not have specific aminotransferase. How is nitrogen from Arg eliminated? Serine A. Dehydratation and deamination H,0 Ser HoO "OOC-OCH2 I NH3+ B. Conversion to glycine H4folate Hjfolate Ser Gly NH3 >- Pyruvate C02 + NH3 C. Decarboxylation Ser -----»----»—Choline CO, D. Conversion to 3-P-glycerate (important for gluconeogenesis) Glu NADH+ 11* NAD* ATP ADP Ser ^ ..... ^ >....... X A. , 3-P-Glycerate transamination P, YUO ... Glu ^ NAD ;| ^— PhosDhoserine < ^— t ' ^ Phosphoserine ■ NADH + II* Glucose The reaction is important for glucose synthesis, the reaction proceeding in opposite direction is the main way of serine synthesis. Glycine A. The main catabolic pathway h^folate Gly - V ^ - C02 + NH3 NAD+ NAD H + H + B. Conversion to serine V J* Gly _ *- Ser --..........Pyruvate C. Side pathway of catabolism nad* nadh + h+ nh3 y—^-*" Oxalate Glyoxylate °2 H2°2 - x > Formiate co2 X ... primary hyperoxaluria, calcium oxalate urolithiasis Threonine -».-*■ Acetyl-CoA Thr Gly A4 Methionine Cystathionine Cys CoASH --==——*■ Propionyl-CoA J Carboxylation B19 ^-''—'2 r Isomeration Succinyl-CoA 8. Hyperhomocysteinemia is a consequence of B|2 vitamin deficiency. What other compound will be formed in the increased amount and what other compound will be missing at B,2 deficiency? 9. What metabolite is accumulated in blood at methylene tetrahydrofolate reductase deficiency? 10. Met does not have specific aminotransferase. How is nitrogen from Met eliminated? H20 Homoserine 2-Oxobutyrate 45 Cysteine A. The main catabolic pathway Cys Oo Cysteine sulfinate Sulfinylpyruvate HSof Pyruvate Sulfite oxidase so|" 2 ATP PAPS Taurine B. Side pathway of catabolism Cys Transamination Mercaptopyruvate SH" ± Pyruvate 11. What amino acids are necessary for conjugation reaction of bile acids? 12. What cofactor is a component of sulfite oxidase? 13. Why cysteine and phenylalanine are not essential when the intake of methionine a tyrosine is sufficient? 14. What is the role of taurine in metabolism? 15. What is significance of PAPS in metabolism? 16. What compound is formed by decarboxylation o cysteine? What cofactor includes this compound in its structure? 46 Selenocysteine It arises co-translationally from serine charged on selenocysteine t-RNA. Seryl-tRNA is converted to the selenocysteyl-tRNA in the reaction with selenophosphate. In the presence of a specific elongation factor that recognizes selenocysteyl-tRNA can be incorporated into proteins. The codon for its recognition is UGA that normally signals STOP. Formation of selenophosphate Se" + ATP-► AMP + Pi + ................................. Selenophosphate Examples of enzymes containing selenocysteine: thioredoxin reductase glutathione peroxidase deiodinase 17. What is the function of glutathione peroxidase in the cells? Aspartate A. Transamination ast Asp B. Urea cycle ATP Asp + Citnilline C. Decarboxylation Asp C02 47 Asparagine Deamidation Asn + . NH3 1_ Glutamate A. Transamination Oxaloacetate Glu B. Oxidative deamination NAD+ NADH+ H+ Glu \ ^ ....... C. Decarboxylation C02 Glu ^ > .......... HoO H,0 ~Xr ................... ATP ADP + P Glutamine Glutamine synthetase B. Deamidation H-.0 .................. \ /» ...................... Glutaminase Glutamine A. Synthesis 48 Proline A. Catabolism NAD(P) NAD(P)H + H Pro a'-Pynoline-5-carboxylate NAD+ NADH + H+ H20 AwZ Glutamate-Y-semialdehyde Glu B. Hydroxylation of proline 2-Oxoglutarate 4-HydroxyPro 18. What is the general name of the enzyme catalyzing the proline hydroxylation? 19. Proline does not have specific aminotransferase. How is nitrogen from Pro eliminated? Histidine A. Catabolism NH3 His ± Hislidase 2 H20 h„folate Glu Urocanate a^-Formiminoplutamate "FIGLU" B. Decarboxylation His C02 J_ 20. What heterocycle is comprised in histidine structure? 21. What cofactor is a component of histidine decarboxylase? 22. Histidine load serves as a test of tetrahydrofolate deficiency. Formiminoglutamate (FIGLU) is excreted into the urine when tetrahydrofolate is deficient. Explain. 23. His does not have specific aminotransferase. How is nitrogen from His eliminated? 49 Leucine Isoleucine Valine Transamination Oxidative decarboxylation V_^C02 = = © 2,3-Dehydrogenation FAD ► FADH2 ATP ADP C02 Carboxylation at C4 i H20 HoO Hydratation on the double bond 3-hydroxy-3-metliylglularyl-CoA (HMG-CoA) Acetoacetate Acetyl-CoA NAD NADH+ IT CoA-SH CoA-SH Propionyl-CoA Acetyl-CoA Carboxylation B12 CoA-SH-^ Isomeralion W2> Succinyl-CoA NAD+ NADH + H+ NAD+ NADH+ H+ Propionyl-CoA Carboxylation B12 Isomeration Succinyl-CoA © (Deficiency of branched-chain 2-oxoacid dehydrogenases) Ketonuria with occurrence of branched chain 2-oxoacids in urine (an inborn error of metabolism called "maple syrup urine disease") 24. What is the cause of methylmalonyl aciduria? Metabolism of what amino acids can display this defect. 50 Lysine A. Catabolism HOOC—CH-(CH2)4- NH2 Lysine -NH2 + 0=C— (CH2)2- | COOH t 2-Oxoglutarate -COOH Allysine + Glu NAD ►NADH + H + ■2-Oxoadipate- 2 CO. ± 2 Acetyl-CoA 2-Aminoadipate Transamination B. Conversion to carnitine Met Met Met Lys -». -». -». ..................................... Trimethyllysine I I Carnitine C. Formation of cross-bridges in collagen Lys Lysyl oxidase (Cu2+) Oondenzation 02 Oxidative deamination Allysine Cross-bridges H202 NH3 25. Lysine does not have specific aminotransferase. How is nitrogen from Lys eliminated? Phenylalanine A. Catabolism Tyrosine 02 H20 Phe ">|{ Tyr 1of* Glu uZ Transamination NADP+ NADPH + H+ COOH AH2 A OH Homogentisate 0, Acetoacetate + Fumarate CS Maleinyl acetoacetate Fumaroyl acetoacetate 26. What is the general name of an enzyme that catalyzes the phenylalanine and tyrosine hydroxylation? 27. What compound is H-donor in the process homogentisate formation? 28. What products are accumulated and consequently excreted in urine when enzyme phenylalanine hydroxylase is deficient? What is the name of this disease? B. Conversion to catecholamines O, H70 Tyr Z DOPA vit. C Dopamine Norepinephrine Epinephrine 29. In which type of reaction is DOPA converted to dopamine? 30. What compound is formed from dopamine by the action of dopamine-P-hydroxylase? C. Conversion to thyroidal hormones 21 iy-- .................................. 3,5-Diiodotyrosine (DIT) 57. Thyroxine (T4) D. Conversion to skin pigments 02, Cu o2, Cu TyT -[J-*■ DOPA -*■ Dopachinone -*■-»- Melanins 0 Disturbances in phenylalanine and tyrosine metabolism Name of the disease Enzyme disturbance Products that accumulates in urine Symptoms Hyperphenylalaninemia type I (phenylketonuria) 1 t Phe, phenylpyruvate, phenylacetate, phenyllactate phenylacetylglutamine, Mental retardation, seizures, psychoses, mousy odour Hypertyrosinemia type II 2 ŤTyr Alkaptonuria 3 T Homogentisate Black urine Hypertyrosinemia type I 4 Liver failure, early death Albinism 5 Absence of skin and eye pigments Enzyme defects: [TJ Phenylalanine hydroxylase, {2} Tyroxine transaminase, fj^ Homogentisate oxygenase [T| Fumaroyl acetoacetate hydrolase, fjT] Tyroxine hydroxylase 31. Explain the formation of phenylalanine catabolites at phenylketonuria. 32. The symptom of alkaptonuria is black urea. Try to explain, what the cause of the dark colour is. 53 Tryptophan A. Catabolism and nicotinamide formation Trp O, CH,—CH—COOH % NH2 O NH—C /V-Formylkynurenine NAD+ H4 folate förmyl-H4 folate CH2—CH—COOH t I \ NH2 O CONH, Nicotinamide NH2 Kynurenine 3-Hydroxykynurenine B, Ala COOH 3 % OH ■*- 3-Hydroxyanthranilate I 97% B. Decarboxylation C02 Trp — 2 Acetyl-CoA 2-Oxoadipate C. Conversion to melatonin HoO Tip 02 n2' 5-Hydroxytryptophan Melatonin Serotonin (A'-acetyl-O-melhyl serotonin) 33. The clinical syndrome resulting from nicotinic acid deficiency is called pellagra. Explain, why this deficiency is widespread within maize-eating areas. 54