Lecture 17: Nitrogen metabolism 1. Urea cycle detoxification of NH 3 2. Amino acid degradation

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1 Lecture 17: Nitrogen metabolism 1. Urea cycle detoxification of NH 3 2. Amino acid degradation Reference material Biochemistry 4 th edition, Mathews, Van Holde, Appling, Anthony Cahill. Pearson ISBN: Lehninger Principles of Biochemistry 4 th edition, David L. Nelson, Michael M. Cox. W. H. Freeman ISBN: Degradation of amino acids Proteases Amino acids are converted into central metabolism for energy generation 國立交通大學生物科技學系蘭宜錚老師 1

2 First step to amino acid degradation removal of amino group Amino acid degradation usually begins with conversion to the corresponding a keto acid by transamination or oxidative deamination. transaminase αkg Glu Conversion of α keto acid to central metabolism NH 3 + NAD(P)H Ammonia is produced as product L amino acid oxidase + NH 3 Transamination as the most flexible reaction for amino acids Transamination is the reversible transfer of an amino group from an a amino acid to an a keto acid, with pyridoxal phosphate as a coenzyme. The equilibrium constants of transamination reactions are close to 1. therefore, the direction of the reaction is dependent on the intracellular concentration of the substrates and products Transamination is involved in both degradation and biosynthesis of amino acids. Glutamate is the most typical/common amino donor as glutamate dehydrogenase is the most important NH3 assimilation enzyme. 國立交通大學生物科技學系蘭宜錚老師 2

3 Transamination facilitated by pyridoxal phosphate (PLP) Transamination: Vitamin B 6 is also called pyridoxine. The active coenzyme has been oxidized to an aldehyde and the hydroxymethyl group at position 5 is phosphorylated. Pyridoxal phosphate (PLP) is the predominant coenzyme form Pyridoxamine phosphate (PMP) is an intermediate form in transamination reactions. Transamination facilitated by pyridoxal phosphate (PLP) PLP is attached to a lysine residue in the active site of the enzyme via a Schiff base (As drawn in the book) Rotate the molecule Schiff base formation 國立交通大學生物科技學系蘭宜錚老師 3

4 Transamination Mechanism Amino group on amino acid substrate attacks Schiff base carbon, replacing the lysine amino group Lysine amino group deprotonates the amino acid α carbon. The electrons go onto Schiff base and rest of the π system, eventually reaching N atom in pyridoxal ring. This carbanion is very well stabilized by resonance into the π system and N atom Transamination Mechanism Electron from the Pyridoxal is used to pull proton from protonated lysine. H 2 O comes in to replace Schiff base, leaving the amino group on PLP. Keto acid is formed. Another keto acid comes in to form Schiff base. Then following step 1 (except now using the amino group on lysine), amino acid is liberated 國立交通大學生物科技學系蘭宜錚老師 4

5 Transaminase detection as a diagnostic tool for organ problems Most aminotransferases use glutamate/a ketoglutarate as one of the two a amino/aketo acid pairs involved. Two such enzymes are important in the clinical diagnosis of human disease serum glutamate oxaloacetate transaminase (SGOT) and serum glutamate pyruvate transaminase (SGPT): These enzymes, abundant in heart and in liver, are released from cells as part of the cell injury that occurs in myocardial infarction, infectious hepatitis, or other damage to either organ. Assays of these enzyme activities in blood serum can be used both in diagnosis and in monitoring the progress of a patient. Very commonly used to check for Liver problems PLP is also used in amino acid racemase & decarboxylase Transamination: Racemation: (Racemase) Decarboxylation: (Decaboxylase) 國立交通大學生物科技學系蘭宜錚老師 5

6 Amino acid racemases Almost all amino acids are made as L amino acids. Many bacteria produce significant amounts of D amino acids (D Ala, D Glu) as part of their peptidoglycan layer. Therefore, bacterial amino acid racemases are also targets for antibiotics. Amino acid decarboxylases Amino acid decarboxylases (different kinds) participate in many different metabolic pathways. Production of many neutral transmitters (e.g. GABA, dopamine, serotonin, histamine, etc.) Bacterial degradation of amino acids. Cadaverine & putrescine are diamines formed from lysine and ornithine. These diamines are used to produdce Nylon. 國立交通大學生物科技學系蘭宜錚老師 6

7 Ammonia (NH 3 ) needs to be excreted Although ammonia is involved in both synthesis and degradation of amino acids, its abnormal accumulation is toxic. Animals have evolved pathways to detoxify NH3. Birds & insects evolved to convert NH3 into uric acid, which is quite insoluble and can be excreted. Uric acid biosynthesis occurs in purine biosynthesis. It contains 4 nitrogen atoms per molecule Most mammals convert NH3 to urea, which is highly soluble, but no ionizable groups (which does not affect ph). It contains 2 nitrogen atoms per molecule. It is produced through Urea cycle. The first biological cycle discovered (also by Hans Krebs and before TCA cycle) Urea is produced in the level. Then transported to the kidneys for excretion Urea cycle Urea cycle was discovered in 1932, 5 years before TCA cycle was discovered by the same person Hans Krebs. Urea cycle uses energy to convert NH 3 (or NH 4+ as dissolved) to urea. The net reaction is: Where fumarate can convert to aspartate with the following net reaction: Fumarate + NH 4+ Asparate Therefore the overall net reaction in terms of energetics (ignoring H2O and converting AMP to ADP) would be: CO NH ATP Urea + 4 ADP + 4 P i 國立交通大學生物科技學系蘭宜錚老師 7

8 Urea cycle carbamoylphosphate synthetase Ornithine transcarbamoylase Argininosuccinate synthetase Urea cycle is composed of mainly 5 enzymes: 1. Carbamoyl phosphate synthetase 2. Ornithine transcarbamoylase 3. Argininosuccinate synthetase Arginase Argininosuccinate synthetase 4. Argininosuccinase Argininosuccinase 5. arginase Conversion of fumarate to asparate Fumarate is generated from urea cycle. However, fumarate can be used to capture another NH 3 in the mitochondria through TCA cycle to OAA, which then undergoes a transamination with glutamate (formed by reductive amination of αkg and NH 3 ) Mitochondria fumarase Malate dehydrogenase 國立交通大學生物科技學系蘭宜錚老師 8

9 Ornithine transcarbamoylase Aspartate add to citrulline replacing carbonyl Oxygen with aspartate Nitrogen Regulation of Urea cycle Animals have long term and short term mechanisms to regulate flux through the urea cycle. Long term: the enzymes of urea cycle are produced in high levels in animals with high protein diet. (low levels if the animal is fed protein free diet) Short term: Carbamoyl P synthetase is allosterically activated by N acetylglutamate, which is made from glutamate + acetyl CoA. Glutamate level is representative of cell s ammonia level, as the one of the first steps of amino acid degradation is transamination to glutamate. Carbamoyl P synthetase is also regulated by covalent modification inactivation of specific lysine residue. However the details of this mechanism is not completely understood yet. 國立交通大學生物科技學系蘭宜錚老師 9

10 NH 3 transport Transport of ammonia to the liver for urea synthesis. The carrier is glutamine in most tissues but is alanine in muscle. Amino acid degradation 國立交通大學生物科技學系蘭宜錚老師 10

11 Essential and non essential amino acids Mammals can degrade all amino acids, however cannot synthesize all amino acids. Those that cannot be synthesized have to come from diet/food. 20 different amino acids = 20 different chemicals = at least 20 different pathways required to metabolize them all. 國立交通大學生物科技學系蘭宜錚老師 11

12 Amino acid degradation Through evolution, there may be more than 1 pathway which exists for degradation of a particular amino acid. These pathways differ in: The organisms which they appear in, The conditions that they are most compatible with, and The resulting central metabolites they produce. Here we will only discuss some of the pathways for amino acid degradation with particular emphasis on how and where (in central metabolism) these amino acid is converted to. Particularly, we focus on the central metabolites Oxaloacetate Pyruvate α Ketoglutarate Glucogenic Succinyl CoA Fumarate Acetyl CoA & acetoacetyl CoA Ketogenic Amino acids degrading to oxaloacetate Aspartate and asparagine are metabolites that are directly related to oxaloacetate through transamination and amide formation. Therefore, degradation of asparagine and aspartate yields oxaloacetate, which can be used directly to gluconeogenesis Asparagine Aspartate 國立交通大學生物科技學系蘭宜錚老師 12

13 Amino acids degrading to Pyruvate 6 amino acids can degrade to form pyruvate. Some amino acids degrade to more than 1 central metabolite (for example, threonine produces acetyl CoA & pyruvate using this pathway) Threonine Glycine Serine Alanine Cysteine Tryptophan Rest of tryptophan degrade to acetyl CoA Glycine and Serin Metabolism Involves Tetrahydrofolate (THF) THF is an important ONE carbon Carrier in the cell (used to generate Methionine, which is used to make SAM) THF participates in metabolism of Serine, glycine, methionine, and histidine. As well as formation of purines 國立交通大學生物科技學系蘭宜錚老師 13

14 THF biosynthesis from folate dihydrofolate reductase dihydrofolate reductase Vitamin B9 THF biosynthesis in bacteria is an antibiotics target Before World War II, one of the few effective antibacterial drugs available was sulfanilamide, one of the class of sulfonamide drugs ( sulfa drugs ). There is a structural similarity between sulfanilamide and p aminobenzoate (PABA), which was known to be essential for bacterial growth. sulfanilamide acts by blocking the normal utilization of PABA. The enzyme incorporating PABA into folic acid is inhibited by sulfonamides. PABA is not required for growth of animal cells, so the drug is not toxic to human cells. Because animal cells do not carry out the synthetic pathway but instead take up fully formed folate from the diet, they are not harmed by the drug 國立交通大學生物科技學系蘭宜錚老師 14

15 Amino acids degrading to α ketoglutarate Part of urea cycle Glutamate Glutamine Histidine Proline Arginine Amino acids degrading to succinyl CoA 4 amino acids can degrade to form succinyl CoA, through propionyl CoA.Recall that propionyl CoA to succinyl CoA conversion from α oxidation. Methionine Threonine Valine Isoleucine 國立交通大學生物科技學系蘭宜錚老師 15

16 Branched chain amino acids Isoleucine, valine, & leucine To degrade these amino acids, they are first converted to corresponding keto acids, which can then undergo branched chain α keto acid dehydrogenase (similar to pyruvate dehydrogenase). Then, reaction sequences similar to β oxidation are used to form acetyl CoA, or other acyl CoA. Methionine degradation How to remove this methyl group? Methionine is converted to SAM, which is used to methylate (or donate methyl group) to another molecule X, yielding SAHC, which can be converted to homocysteine. Homocysteine can then convert to methionine in its biosynthesis using methyl group from methyl THF 國立交通大學生物科技學系蘭宜錚老師 16

17 Amino acids degrading to Acetyl CoA Saccharopine pathway 7 amino acids can degrade to form acetyl CoA. Tryptophan Lysine Phenylalanine Tyrosine Leuccine Isoleucine Threonine (not shown here) Lysine degradation Oxidize this to acid Transaminate this to keto group Acetyl CoA 國立交通大學生物科技學系蘭宜錚老師 17

18 Tryptophan degradation Tryptophan is an important precursor to NAD+ biosynthesis Phenylalanine & Tyrosine degradation 國立交通大學生物科技學系蘭宜錚老師 18

19 Human genetic disorders associated with amino acid metabolism 國立交通大學生物科技學系蘭宜錚老師 19