1. Derivatives of Amino Acids and Metabolism of Nucleotides
CH353 January 29, 2008

2. Anabolic Role of the Citric Acid Cycle
Error:
glycine not glutamate provides carbons for purines
Purines X
Biosynthesis of amino acids & derivatives from citric acid cycle intermediates require anaplerotic reactions (red arrows) for replenishing metabolites

3. Derivatives of Amino Acids
Porphyrins and Heme
Glycine + Succinyl-CoA (animals)
Glutamate (bacteria & plants)
Non-ribosomal peptide synthesis
peptidoglycan, antibiotics
glutathione (glutamate + cysteine + glycine)
Modified amino acids
plant compounds, neurotransmitters, polyamines
Nucleotide heterocyclic bases
purines and pyrimidines

4. Biosynthesis of Heme
animals
heme precursor
bacteria, plants

5. Biosynthesis of Heme

6. Genetic Deficiencies in Heme Biosynthesis

7. Catabolism of Heme purple Regulated step: 3 isozymes green yellow
Important serum antioxidant
Bile pigment
yellow (oxidized)
red-brown (reduced)

8. Reactions with Monooxygenases
Use 2 reductants for O2 (mixed-function oxygenases)
One reductant accepts an O atom
Other reductant provides 2 H’s to the second O atom
General Reaction:
AH + BH2 + O–O → A–OH + B + H2O

9. Biosynthesis of Nitric Oxide
NO involved in intercellular signaling
NO synthase (a mixed-function oxygenase)
dimer, similar to NADPH cytochrome P450 reductase
cofactors: FMN, FAD, tetrahydrobiopterin, Fe3+ heme
catalyzes a 5 e- oxidation

10. Biosynthesis of Creatine
metabolite for storage of high energy transfer potential phosphate
phosphorylated at high [ATP]
amidinotransferase exchanges amino acids
glycine for ornithine
1 substrate and 1 product same as for arginase reaction except different amidino group acceptor
glycine instead of water
S-adenosylmethionine methyl donor

11. Biosynthesis of Glutathione
reducing agent (redox buffer)
non-ribosomal peptide synthesis
carboxyl groups activated with ATP (acyl phosphate intermediates)

12. Non-ribosomal Peptide Synthesis
Microbial peptides are synthesized by multi-modular synthases; similar to fatty acid biosynthesis
Modular complexes of enzymes for recognition, activation, modification and condensation of a specific amino acid to the growing polymer
Features use of unusual amino acids, D-enantiomers, and non-α peptide bonds
Peptidoglycans, antibiotics and ionophores

13. Reactions with Pyridoxal Phosphate
Amino acid racemase reactions
L-alanine ↔ D-alanine
Inhibitors of alanine racemase:
Antibiotics – peptidoglycan biosynthesis

14. Biosynthesis of Plant Compounds
phenylalanine, tyrosine, tryptophan precursors for plant compounds:
lignin (phenolic polymer)
indole-3-acetate (auxin)
tannins
alkaloids, e.g. morphine
flavors, e.g. cinnamon, nutmeg, cloves, vanilla, cayenne pepper

15. Reactions with Pyridoxal Phosphate
Decarboxylase reactions
Histidine → Histamine + CO2
Ornithine → Putrescine + CO2

16. Biosynthesis of Neurotransmitters
Pathways involve decarboxylases and mixed-function oxygenases (monooxygenases)

17. Biosynthesis of Spermidine and Spermine
Pathway involves decarboxylases and S-adenosylmethione alkylation

18. African Sleeping Sickness
Caused by Trypanosoma brucei rhodesiense
Vaccines are ineffective: repeated change of coat antigen
Therapy based on inhibitor of polyamine biosynthesis

19. Mechanism of Ornithine Decarboxylase

20. Inhibition of Ornithine Decarboxylase
DMF-Ornithine

21. Study Problem
Antihistamines are compounds that block histamine synthesis or binding to its receptor
Histamine is synthesized from histidine by a pyridoxal phosphate dependent decarboxylase
Design an antihistamine drug candidate, based upon the mechanism for decarboxylation
Show the structure and its proposed mechanism of action

22. Overview of Nucleotide Metabolism
Nucleotide functions
Activated precursors for synthesis of RNA, DNA and cofactors
Activation of biosynthetic precursors
Energy for cellular processes
Signal transduction
Biosynthetic pathways
de novo synthesis of purines and pyrimidines
differ in order of attachment of ribose to base
salvage pathways
reacting a base with activated 5-phosphoribose (PRPP)

23. Precursors for Nucleotide Biosynthesis
5-phosphoribosyl-1-pyrophosphate
ribose phosphate pyrophosphokinase
Ribose 5-phosphate + ATP → 5-phosphoribosyl-1-pyrophosphate + AMP

24. Precursors for Nucleotide Biosynthesis
Tetrahydrofolate (H4 folate) derivatives
N5,N10-methylene-H4 folate
thymidylate biosynthesis
N5-formyl-H4 folate
purine biosynthesis

25. Precursors for Nucleotide Biosynthesis
Amino Acids
Glycine for purine biosynthesis
Aspartate for pyrimidine biosynthesis
Amino Acid Nitrogen
α-amino group of aspartate (purines)
aspartate + [acceptor] + ATP → succinyl-amino-[acceptor] + ADP + Pi
succinyl-amino-[acceptor] → amino-[acceptor] + fumarate
amide group of glutamine (purines, pyrimidines)
glutamine + [acceptor] + ATP → amino-[acceptor] + glutamate + ADP + Pi

26. Activation of Amino Acceptors
carboxylate or carbonyl acceptor are activated with ATP
acyl-phosphate or phospho-enol intermediates formed
nucleophilic substitution of phosphate with amino group
R–C–O– O
R–C–OPO3–2 O
R–C–NHR’ O
ATP
ADP
R’NH2
PO4–2
C–C–R O H
C–C–R
OPO3–2
C–C–R
NHR’

27. Biosynthesis of the Purine Ring
Multi-step synthesis from many precursors
(numbers indicate order of addition to purine ring from PRPP) 1 2 3 4 5 6 7

28. Purine Biosynthesis glutamine-PRPP amidotransferase GAR synthetase
glutamine donates amide nitrogen to activated 5-phosphoribose (PRPP)
committed step for purine synthesis
product unstable t½ = 30 seconds
GAR synthetase
glycine carboxyl activated with ATP
Pi displaced; amide bond formed
GAR transformylase
N10-formyl tetrahydrofolate donates formyl group to glycine amino group
FGAR amidotransferase
ATP activates carbonyl group
amidotransfer displaces Pi

29. Purine Biosynthesis FGAM cyclase (AIR synthetase) in bacteria & fungi:
ATP activates carbonyl
cyclization of imidazole ring
in bacteria & fungi:
N5-CAIR synthetase
ATP activates HCO3-
carbamoylation of exocyclic amine
N5-CAIR mutase
transfer of carboxylate to ring
in higher eukaryotes:
AIR carboxylase
formation of only C-C bond
no cofactors or ATP required

30. Purine Biosynthesis SAICAR synthetase SAICAR lyase
aspartate is amino donor
ATP activates carboxylate
aspartate amino replaces Pi
SAICAR lyase
fumarate is eliminated
steps 8 & 9 analogous to urea cycle
AICAR from histidine biosynthesis
AICAR transformylase
N10-formyl H4 folate donates formyl group to glutamine-derived amine
IMP synthase
cyclization of second purine ring
ATP activation not required

31. Organization of Purine Biosynthetic Enzymes
Purine biosynthesis organized into multienzyme complexes
In eukaryotes, multifunctional proteins for:
Steps 1, 3 & 5
Steps 6a & 8
Steps 10 & 11
In bacteria, separate enzymes associate in large complexes
Channeling of intermediates avoids dilution of reactants

32. Synthesis of Adenylate and Guanylate
AMP synthesis uses GTP for activation; amine from aspartate
GMP synthesis uses ATP for activation; amide from glutamine
Reciprocal Regulation:
GTP for needed for AMP synthesis
ATP needed for GMP synthesis

33. Regulation of Purine Biosynthesis in E. coli
Feedback Inhibition (negative)
Inhibition of 1st step in common pathway by IMP, AMP & GMP
Inhibition of 1st step in branch
AMP inhibits AMP synthesis
GMP inhibits GMP synthesis
Inhibition of PRPP synthesis by phosphorylated end products ADP, GDP and others
Reciprocal Regulation (positive)
Requirements of:
ATP for GMP synthesis
GTP for AMP synthesis

34. Nucleotide Biosynthesis
Purine Biosynthesis
Hypoxanthine (a purine) is assembled on the ribose 5-phosphate → Inosinate (IMP)
Precursors:
PRPP
Glycine
H4 folate-formate (2)
HCO3–
Glutamine (amide-N) (2)
Aspartate (amino-N)
IMP → AMP
IMP → XMP → GMP
Pyrimidine Biosynthesis
Orotate (a pyrimidine) is made first then added to ribose 5-phosphate → Orotidylate
Precursors:
Carbamoyl phosphate
HCO3–
Glutamine (amide-N)
Aspartate
PRPP
Orotidylate → UMP → UDP → UTP → CTP

35. Pyrimidine Biosynthesis
Carbamoyl Phosphate Synthetase II
cytosolic CPS II enzyme involved in pyrimidine biosynthesis
mitochondrial CPS I involved in arginine & urea synthesis
bacteria have single enzyme for both functions
Steps:
bicarbonate phosphate synthesis (1st activation)
carbamate synthesis (NH3 from glutamine hydrolysis)
carbamoyl phosphate synthesis (2nd activation)

36. Carbamoyl Phosphate Synthetase
Bacterial enzyme has 2 subunits (blue & grey) with 3 active sites joined by a substrate channel (yellow wire mesh)
1st site: Glutamine releases NH4+ (glutamine in green)
2nd site: HCO3– is phosphorylated with ATP and reacts with NH4+ to form carbamate (ADP in blue)
3rd site: Carbamoyl phosphate is synthesized by phosphorylating carbamate with ATP (ADP in red)

37. Pyrimidine Biosynthesis
aspartate transcarbamoylase
activated carbamoyl group transferred to amine group of aspartate
Pi displaced; amide bond formed
committed step in pyrimidine synthesis
dihydroorotase
cyclization of pyrimidine ring
dihydroorotate dehydrogenase
oxidation of C-C bond using NAD+
orotate phosphoribosyl transferase
pyrimidine ring (orotate) is transferred to activated 5-phosphoribose (PRPP)
PPi lost; aminoglycan bond formed
analogous to pyrimidine salvage

38. Pyrimidine Biosynthesis
orotidylate decarboxylase
catalyzes synthesis of UMP
very efficient enzyme
uridylate kinase
nucleoside monophosphate kinase specific for UMP
nucleoside diphosphate kinase
generic enzyme for (d)NDP’s
cytidylate synthetase
an amidotransferase
UTP is aminated using glutamine
carbonyl group is activated with ATP to form acyl phosphate intermediate
Cytidine 5’-triphosphate (CTP)

39. Pyrimidine Biosynthesis Enzyme Complexes
Eukaryotes have a multifunctional protein with the first 3 enzymes in pyrimidine biosynthetic pathway
C carbamoyl phosphate synthetase II
A aspartate transcarbamoylase
D dihydroorotase
CAD has 3 identical polypeptides (Mr 230,000) each with sites for all 3 reactions

40. Regulation of Pyrimidine Biosynthesis
Feedback inhibition of 1st step aspartate transcarbamoylase (ATCase) by CTP
Bacterial ATCase has:
6 catalytic subunits
6 regulatory subunits
Allosteric inhibition:
2 conformations of ATCase: active ↔ inactive
binding of CTP to regulatory subunits shifts conformation active → inactive
ATP reverses effect of CTP

41. Activation of Nucleotides
Nucleoside monophosphate kinases
specific enzyme for each base (e.g. adenylate kinase)
nonspecific for ribose (ribose or 2’-deoxyribose)
ATP + NMP ADP + NDP
Nucleoside diphosphate kinase
generic enzyme, nonspecific for base or ribose
nonspecific for phosphate donor or acceptor
NTP + NDP NDP + NTP
donor acceptor acceptor donor

42. Nucleotides for DNA Synthesis
2 Modifications:
ribonucleotides reduced to 2’-deoxyribonucleotides
NDP → dNDP
uracil (uridylate) methylated to thymine (thymidylate)
dUMP → dTMP

43. Reduction of Nucleotides
NDP is reduced to dNDP by reduced form of ribonucleotide reductase
Oxidized form of ribonucleotide reductase is reduced by either glutaredoxin or thioredoxin
Oxidized form of glutaredoxin is reduced by glutathione
Oxidized form of thioredoxin is reduced by FADH2
Oxidized glutathione and FAD are reduced by NADPH

44. Regulation of Ribonucleotide Reductase
Ribonucleotide Reductase (E. coli)
Active sites are between each R1 and R2 subunit
Two R2 subunits each contain a tyrosyl radical and a binuclear Fe3+ cofactor
Two R1 subunits each have sites for enzyme activity and substrate specificity
The (d)NTP bound to substrate specificity sites determines which NDP is reduced to dNDP

45. Regulation of Ribonucleotide Reductase
Binding at activity regulatory sites:
ATP activates enzyme
dATP inhibits enzyme
Binding at substrate specificity sites:
ATP or dATP: ↑dCDP ↑dUDP
dTTP: ↑dGDP ↓dCDP ↓dUDP
dGTP: ↑dADP ↓dGDP ↓dCDP ↓dUDP

46. Biosynthesis of Thymidylate
Precursors for thymidylate (dTMP) synthesis may arise from (d)CTP or (d)UTP pools
CTP
UTP
nucleoside diphosphate kinase
UMP
cytidylate synthetase
uridylate kinase

47. Cyclic pathway for conversion of dUMP to dTTP
Thymidylate synthase uses N5,N10-Methylene-H4 folate as both one-carbon source and reducing agent
Dihydrofolate reductase reduces H2 folate → H4 folate with NADPH
Serine hydroxymethyl transferase reaction restores N5,N10-Methylene-H4 folate
Net reaction:
dUMP + NADPH + serine →
dTMP + NADP+ + glycine

48. Chemotherapeutic Agents
Inhibitors of glutamine amidotransferases:
Block purine & pyrimidine biosynthesis
Inhibitors of thymidylate synthesis:
thymidylate synthase
dihydrofolate reductase

49. Chemotherapy Targets

50. Group Study Problem
Conversion of dUTP to dTTP by thymidylate synthase requires N5,N10-Methylene-H4 folate as both one-carbon source and reducing agent
N5,N10-Methylene-H4 folate and glycine are produced in a reversible reaction whereby the hydroxymethyl group of serine in transferred to H4 folate
What effect may an elevated glycine:serine ratio during photorespiration have on DNA synthesis?
January 31, 2008

51. Catabolism of Purine Nucleotides
Adenosine deaminase deficiency:
severe immunodeficiency disease; loss of T- and B-cells
100x ↑ dATP (inhibitor of ribonucleotide reductase) ↓ dNTP’s, ↓ DNA synthesis
Catabolism produces purine bases for salvage pathways
Uric acid
catabolic end product in humans
gout – accumulation of uric acid in joints and urine
treatment with xanthine oxidase inhibitors, e.g. allopurinol

52. Purine Catabolism Pyrimidine Catabolism

53. Salvage Pathways for Nucleotides
de novo biosynthesis of purine nucleotides assembles the purine ring on 5’-phosphoribose
Salvage pathway adds completed purine base to PRPP
Adenosine phosphoribosyltransferase
Adenine + PRPP → AMP + PPi
Hypoxanthine-guanine phosphoribosyltransferase
Hypoxanthine + PRPP → IMP + PPi
Guanine + PRPP → GMP + PPi
Lesch-Nyhan syndrome:
deficiency in hypoxanthine-guanine phosphoribosyltransferase

54. Biosynthesis of Cofactors
Nicotinamide Adenine Dinucleotide (NAD)
PRPP
PPi
ATP
PPi
Gln
Glu
Nicotinate (Niacin)
Nicotinate ribonucleotide
Desamido NAD+
NAD+
Flavin Adenine Dinucleotide (FAD)
ADP
ATP
ATP
PPi
Riboflavin
5’-phosphate
Riboflavin
FAD