1. Pratt and Cornely Chapter 18
Nitrogen Metabolism
Pratt and Cornely Chapter 18

2. Overview Nitrogen assimilation Amino acid biosynthesis
Nonessential aa
Essential aa
Nucleotide biosynthesis
Amino Acid Catabolism
Urea Cycle
Juicy Steak Part 2

3. Nitrogen fixation
Bacteria
Nitrogenase
Costly—16 ATP per N2 molecule

4. Assimilation into Amino Acids
In microorganisms/plants: assimilation of ammonia is key—synthesis of most amino acids
Glutamine synthetase incorporates amino group
Coupled to glutamate synthase: reductive amination of a-ketoglutarate to glutamate

5. Net Reaction a-KG + NH3 +ATP + NADPH  Glu + ADP + NADP+
Ultimately, this is incorporation of ammonia into an amino acid
Glutamate can then be used to distribute nitrogen into other amino acids (transamination)

6. Assimilation into Amino Acids
In humans: acquire nitrogen in amino acids
Amino acids in diet
Glutamate distributes amino group to new amino acids through transamination
No need for glutamate synthase
Glutamine synthetase used for different purpose: to “mop up” ammonia

7. Transamination
Transfers assimilated nitrogen into all other amino acids
Requires PLP cofactor

9. Biosynthesis Dietary consideration Ambiguous
Stage of life (Arg)
Precursor (Tyr, Cys)
Mechanism of biosynthesis can be grouped

10. Amino Acid Biosynthesis* * * * * * * * * * *

11. Non-essential Amino Acid Biosynthesis
Transamination
Pyruvatealanine
Oxaloacetateaspartate
a-ketoglutarateglutamate
Amidation
Glutamine (glutamine synthetase)
Asparagine (asparagine synthetase)

12. Glutamate Backbone

13. Serine/Glycine 3-phosphoglycerate Serine Serineglycine
THF as a major one-carbon transfer vitamin

15. Cysteine
Serinecysteine by incorporating sulfur from homocysteine (Made from methionine)

17. Phenylalanine Monooxygenase uses O2 for hydroxylation
Other atom ends up in water
Requires a reducing cofactor called tetrahydrobiopterin
Also first step in catabolism of phenylalanine
PKU

18. Neurotransmitters
Which amino acid?

19. Nucleotide Biosynthesis
5-PRPP
Purine: base built onto ribose
Asp, Gly, Glu, THF, bicarbonate
IMP produced

20. AMP/GMP Production Branched pathway AMP: amination GMP: oxidation
Branch allows for reciprocal regulation

21. Pyrimidines Contrast Base made first, then attached to 5PRPP
Not branched: UMP made to UTP then to CTP

22. Ribonucleotide Reductase
Essential reaction: reduction to make dNDP
Very difficult reaction
Free radical
Enzyme is oxidized in the process
Reduced by thioredoxin
In turn, thioredoxin reduced by NADPH

24. Production of TMP dUTP must be converted to TMP quickly
Methylene donated from THF by thymidylate synthase
THF oxidized to DHF
Chemotherapy: dUMP analog

25. Regenerating THF DHF must be reduced to THF by DHF reductase
NADPH dependent
Chemotherapy target
DHF analogs such as methotrexate

26. Catabolism Salvage pathway through phosphorolysis
Purines made into uric acid (waste)
Pyrimidines broken down into catabolic intermediates

27. Amino acid catabolism

28. Ketogenic vs. Glucogenic

29. Problem 35
The catabolic pathways for the 20 amino acids vary considerably, but all amino acids are degraded to one of seven metabolites: pyruvate, a-ketoglutarate, succinyl-CoA, fumarate, oxaloacetate, acetyl CoA, or acetoacetate. What is the fate of each of these metabolites?

31. Amino Acid Degradation
Transamination and deamination
Then carbon chain is metabolized
Examples:

32. Pyruvate Producing
A variety of enzymes convert 3-C chains

33. Glutamate Family
25% of dietary intake

34. Thr: Both Glucogenic and Ketogenic
Many mammals: or
aminoacetone
pyruvate
In humans:

35. Glycine Cleavage system

36. Branched Amino Acids Major energy source in muscle
Steps of degradation
Transamination
Oxidative decarboxylation (Pyruvate DH)
Beta oxidation
Valine: succinyl CoA
Isoleucine: succinyl CoA and acetylCoA
Leucine: acetyl CoA and ketone body

37. Problem 40
Leucine is degraded to acetyl CoA and acetoacetate by a pathway whose first two steps are identical to those of valine degradation (Figure 18-11). The third step is the same as the first step of fatty acid oxidation. The fourth step involves an ATP-dependent carboxylation, the fifth step is a hydration, and the last step is a cleavage reaction to give products. Draw the intermediates of leucine degradation.

39. Aromatic Amino Acids Monoxygenases and Dioxygenases
First recognition of inborn errors of metabolism

40. Problem51
List all the reactions in this chapter that generate free ammonia.

41. Ammonia Processing
Most tissues: glutamine synthetase “mops up” ammonium generated in metabolism
glutamine then sent through blood to liver
Deaminated in liver to give glutamic acid
glutaminase

42. Ammonia Processing Muscle: The alanine-glucose cycle
Glutamate also accepts amino group from other amino acids

43. Glutamate Dehydrogenase
Reversible reaction
Grabs free ammonia and releases it in liver mitochondria

44. Role of Liver Mitochondia
Sequester toxic ammonia
Make less toxic, execrable form
Urea Cycle

45. Carbamoyl phosphate Cost of 2 ATP Activation of ammonia for
Phosphate leaving group
Activation of ammonia for
Excretion
biosynthesis

46. Problem 52
Which three mammalian enzymes can potentially “mop up” excess NH4+?

47. Urea Cycle

48. Chemistry of Urea Cycle
Catalytic ornithine
Fumarate
Urea = 4 ATP
+ ATP
+ AMP

49. Compartmentalization

50. Urea Cycle Regulation Carbamoyl phosphate synthetase
Amino acid catabolism boosts acetyl CoA and glutamate levels
Produces activator

51. Problem 55
An inborn error of metabolism results in the deficiency of argininosuccinase. What could be added to the diet to boost urea production aid in ammonia secretion? (Argininosuccinate can be excreted.)

52. Solving Metabolic Problems
Arginosuccinase deficiency
Low protein diet
Minimize ammonium
High arginine diet
Provide carrier
excreted X

53. Nitrogen Flow Overview

54. Summary: Main Players
Glutamate: in liver, receives nitrogen from AA, then ammonia is released in liver mitochondria
Glutamine: ammonia transport; biosynthesis
Alanine: ammonia transport
Aspartate: nitrogen donor to urea
Arginine: urea cycle