Non protein nitrogen compounds
metabolism
Porphyrins & Nuleobases
1
Heme Metabolism
•Heme biosynthesis
•Heme degradation
2
Biosynthesis of Heme
Production of Aminolevulinic acid from 2 carbon
amino acid glycine and succinyl CoA in the
presence of Ala synthase
Requires two vitamines - pyridoxal phosphate
and pantothenic acid
ALA synthase is an important rate limiting factor
(heme represses - sex hormones enhance - high
glucose blocks)
3
4
Two ALA molecules are joined in the
presence of the enzyme delta
aminolevulinic dehydratase
Forms porphobilinogen
Lead inhibits this step
5
6
Four porphobilinogen molecules
condense to form
hydroxymethylbilane and then
uroporphyrinogen III
Requires porphobilinogen
deaminase (uroporphyrinogen
synthtase) and uroporphyrinogen III
co-synthtase
7
8
Decarboxylation (remove COOH) of
the four acetic acid side chains of
uroporphyrinogen III to form methyl
(CH3)
Forms coproporphyrinogen III
Catabolized by the enzyme
uroporphyrinogen decarboxylase
9
10
Conversion of coproporphyrinogen
III to protoporphyrinogen III
Two propionic acid (CH2-CH2-COOH)
convert to two vinyl (CH2=CH2)
Requires coproporphyrinogen
oxidase and oxygen as a hydrogen
acceptor
Moves heme synthesis back into the
mitochondria
11
12
Fifteen possible isomers of
protoporphyrinogen can form
Normal mitochondrial physiology
leads to the formation of only one of
these isomers (protoporphyrinogen
IX)
Protoporphyrinogen oxidase is
involved in this reaction and oxygen
as a hydrogen acceptor
13
14
15
Heme
A complex of iron and
protoporphyrin (a porphyrin ring)
16
Porphyrins
Protoporphyrin
Coproporphyrin
Uroporphyrin
17
18
COORDINATED REGULATION OF HEME
AND GLOBIN SYNTHESIS:
Heme:
inhibits activity of pre-existing -ALA synthase
diminishes the transport of -ALA synthase from cytoplasm
to mitochondria after synthesis of the enzyme.
represses the production of -ALA synthase by regulating gene
transcription.
stimulates globin synthesis to ensure that levels of free heme
remain low in concentration.
Inhibition of the synthase and stimulation of globin synthesis are
the most important aspects in balancing hemoglobin production.
19
Heme Biosynthesis: Porphyrias
• Cruelly referred to as a Vampire’s disease.
• Can be caused by lead poisoning: The fall
of the Roman Empire!
20
Not a ‘vampire’s’ disease
Some symptoms of porphyrias have lead
people to believe that these diseases
provide some basis for vampire legends:
Extreme sensitivity to sunlight
Anemia
This idea has been discarded both for
scientific reasons:
Porphyrias do not cause a craving for
blood.
Drinking blood would not help a victim
of porphyria.
And for compasionate reasons:Porphyria is a rare, but
21
frightening condition: hard to diagnose and there is no cure.
Mitochondria
PORPHYRIAS
GLYCINE + SuccinylCoA
Agent Orange
ALA synthase
-aminolevulinic acid(ALA)
Porphobilinogen(PBG)
hydroxymethylbilane
uroporphyrinogen III
coprophyrinogene III
Protoporphyrinogene IX
protoporphyrin IX
Heme
3p21/Xp11.21
ALA dehydratase
ALA-dehydratase
Deficiency porphyria
PBG deaminase
Acute intermittent
porphyria
Uroporphyrinogen III
cosynthase 10q26
Congenital erythropoietic
porphyria
Uroporphyrinogen
decarboxylase
Prophyria
cutanea tarda
Coproporphyrinogen
oxidase 9
Herediatary
coproporphyria
Protoporphyrinogen
oxidase 1q14
Variegate
porphyria
Ferrochelatase
Erythropoietic
protoporphyria
9q34
11q23
1q34
18q21.3
22
porphyrias
Type
Enzyme
Involved
Major Symptoms
Acute intermittent
porphyria
Uroporphyrinogen
synthase
Abdominal pain
Neuropsychiatric
urinary porphobilinogen
Congenital
erythropoietic
porphyria
Uroporphyrinogen
cosynthase
Photosensitivity
urinary uroporphyrin
Porphyria cutanea
tarda
Decarboxylase
Photosensitivity
Variegate porphyria
Oxidase
Erythropoietic
protoporphyria
Laboratory tests
porphobilinogen




urinary uroporphyrin
porphobilinogen
Ferrochelatase

Photosensitivity
Abdominal pain
Neuropsychiatric
urinary uroporphyrin
fecal coproporphyrin
fecal protoporphyrin



Photosensitivity
fecal protoporphyrin
red cell protoporphyrin


23
Heme Degradation
NADPH
HEME
O2
NADP+
Fe+3
Heme Catabolism
(opens the porphyrin ring)
BILIVERDIN
NADPH
NADP+
BILIRUBIN
BILIRUBIN diglucuronide
BILE
24
BLOOD
CELLS
Stercobilin
excreted in feces
Urobilin
excreted in urine
Hemoglobin
Globin
Heme
O2
Heme oxygenase
Urobilinogen
formed by bacteria
INTESTINE
KIDNEY
reabsorbed
into blood
CO
Biliverdin IX
via bile duct to intestines
NADPH
Bilirubin diglucuronide
(water-soluble)
Biliverdin
reductase
NADP+
Bilirubin
(water-insoluble)
2 UDP-glucuronic acid
via blood
to the liver
Bilirubin
(water-insoluble)
LIVER
Figure 2. Catabolism of hemoglobin
25
Jaundice
Hyperbilirubinemia:
Two forms:
Direct bilirubin: Conjugated with
glucoronic acid
Indirect bilirubin: unconjugated,
insoluble in water.
26
What’s the cause of jaundice?
1- Increased production of bilirubin by hemolysis or blood disease:
•Increase in blood indirect bilirubin
•Called pre-hepatic jaundice
•Stool color remains normal.
2- Abnormal uptake or conjugation of bilirubin:
•Leads to non-hemolytic unconjugated hyperbilirubinemia
•Increased indirect bilirubin.
•Stool color turns gray.
•Caused by liver damage or disease.
27
3- Cholestasis = Problems with bile flow.
a: Intrahepatic cholestasis: hyper conjugated bilirubinemia
•Increase in blood indirect and direct bilirubin
•Caused by liver damage or disease: eg cirrhosis, hepatitis
•Can also occur in pregnancy:
b:Extrahepatic cholestasis:
•Blockage of bilirubin transport in the bilary tract.
•Increased direct bilirubin.
•Stool color turns gray.
•Caused by: Tumors or gall stones.
28
Examples of hyperbilirubinemia
A. Hemolytic anemia
B. Hepatitis
C. Biliary duct stone
excess
hemolysis
 unconjugated bilirubin
(in blood)
 conjugated bilirubin
(released to bile duct)
 unconjugated bilirubin
(in blood)
 conjugated bilirubin
(in blood)
 unconjugated bilirubin
(in blood)
 conjugated bilirubin
29
(in blood)
Genetic Disorders of Bilirubin Metabolism
Condition
Defect
Bilirubin
Clinical Findings
Crigler-Najjar
syndrome
severely defective
Unconjugated
UDP-glucuronyltransferase bilirubin 
Profound jaundice
Gilberts
syndrome
reduced activity of
Unconjugated
UDP-glucuronyltransferase bilirubin 
Very mild jaundice
during illnesses
DubinJohnson
syndrome
abnormal transport of
conjugated bilirubin into
the biliary system
Moderate jaundice
Conjugated
bilirubin 
30
Nucleotides: Synthesis and
Degradation
31
Roles of Nucleotides
•Precursors to nucleic acids (genetic material and non-protein
enzymes).
•Currency in energy metabolism (eg. ATP, GTP).
•Carriers of activated metabolites for biosynthesis
(eg. CDP, UDP).
•Structural moieties of coenzymes (eg. NAD, CoA).
•Metabolic regulators and signal molecules (eg. cAMP,
cGMP, ppGpp).
32
Nitrogenous Bases
Purines
Pyrimidines
N1: Aspartate Amine
C2, C8: Formate
N3, N9: Glutamine
C4, C5, N7: Glycine
C6: Bicarbonate Ion
33
Purine degredation
AMP deamination in muscle, hydrolysis in other tissues.
Xanthine oxidase:contains FAD, molybdenum, and non-heme iron.
In primates, uric acid is the end product, which is excreted.
34
Purine Nucleotides
• Get broken down into Uric Acid (a purine)
35
Common treatment for gout: allopurinol
Allopurinol is an analogue of hypoxanthine that strongly
inhibits
xanthine oxidase. Xanthine and hypoxanthine, which
are soluble, are accumulated and excreted.
36
Purine degredation in
other animals
37
Uric Acid Excretion
• Humans – excreted into urine as insoluble
crystals
• Birds, terrestrial reptiles, some insects –
excrete isoluble crystals in paste form
(conserve water)
• Others – further modification :
Uric Acid  Allantoin  Allantoic Acid  Urea 
Ammonia
38
Gout
• Impaired excretion or overproduction of uric
acid
• Uric acid crystals precipitate into joints (Gouty
Arthritis), kidneys, ureters (stones)
• Lead impairs uric acid excretion – lead
poisoning from pewter drinking goblets
– Fall of Roman Empire?
• Xanthine oxidase inhibitors inhibit production
of uric acid, and treat gout
• Allopurinol treatment – hypoxanthine analog
that binds to Xanthine Oxidase to decrease uric
acid production
39
Catabolism of
pyrimidines
40
Biosynthetic routes: De novo and salvage pathways
De novo pathways
Almost all cell types have the ability to synthesize purine and
pyrimidine nucleotides from low molecular weight precursors in
amounts sufficient for their own needs.
The de novo pathways are almost identical in all organisms.
Salvage pathways
Most organisms have the ability to synthesize nucleotides from
nucleosides or bases that become available through the diet or from
degredation of nucleic acids.
In animals, the extracellular hydrolysis of ingested nucleic acids
represents the major route by which bases become available.
41
Reutilization and catabolism of purine and pyrimidine bases
blue-catabolism
red-salvage pathways
endonucleases:
pancreatic RNAse
pancreatic DNAse
phosphodiesterases:
usually non-specific
42
Purine Catabolism and Salvage
• All purine degradation in animals leads to uric acid
• Ingested nucleic acids are degraded by pancreatic
nucleases, and intestinal phosphodiesterases in the intestine
• Group-specific nucleotidases and non-specific
phosphatases degrade nucleotides into nucleosides
– Direct absorption of nucleosides
– Further degradation
Nucleoside + H2O  base + ribose (nucleosidase)
Nucleoside + Pi  base + r-1-phosphate (n. phosphorylase)
NOTE: MOST INGESTED NUCLEIC ACIDS ARE DEGRADED AND
EXCRETED.
43
Intracellular Purine Catabolism
• Nucleotides broken into nucleosides by action of
5’-nucleotidase (hydrolysis reactions)
• Purine nucleoside phosphorylase (PNP)
–
–
–
–
Inosine  Hypoxanthine
Xanthosine  Xanthine
Guanosine  Guanine
Ribose-1-phosphate splits off
• Can be isomerized to ribose-5-phosphate
• Adenosine is deaminated to Inosine (ADA)
44
Intracellular Purine Catabolism
• Xanthine is the point of convergence for the
metabolism of the purine bases
• Xanthine  Uric acid
– Xanthine oxidase catalyzes two reactions
• Purine ribonucleotide degradation pathway
is same for purine deoxyribonucleotides
45
PRPP: a central metabolite in de novo and salvage pathways
PRPP synthetase
Enzyme inhinited by AMP, ADP, and GDP. In E. coli, expression is repressed
by PurR repressor bound to either guanine or hypoxanthine.
Roles of PRPP: his and trp biosynthesis, nucleobase salvage pathways, de
novo synthesis of nucleotides
46
Example of a salvage pathway: guanine phosphoribosyl transferase
In vivo, the reaction is driven to the right by the action of pyrophosphatase
Shown: HGPRT, cells also have a APRT.
47
Purine Salvage
• Adenine phosphoribosyl transferase (APRT)
Adenine + PRPP  AMP + PPi
• Hypoxanthine-Guanine phosphoribosyl transferase
(HGPRT)
Hypoxanthine + PRPP  IMP + PPi
Guanine + PRPP  GMP + PPi
(NOTE: THESE ARE ALL REVERSIBLE REACTIONS)
AMP,IMP,GMP do not need to be resynthesized de
novo !
48
De novo biosynthesis of purines: low molecular weight
precursors of the purine ring atoms
49
Synthesis of IMP
The base in IMP is called
hypoxanthine
Note: purine ring built up at
nucleotide level.
precursors:
glutamine (twice)
glycine
N10-formyl-THF (twice)
HCO3
aspartate
In vertebrates, 2,3,5 catalyzed
by trifunctional enzyme,
6,7 catalyzed by bifunctional
enzyme.
50
Pathways from IMP to AMP and GMP
G-1: IMP dehydrogenase
G-2: XMP aminase
A-1: adenylosuccinate
synthetase
A-2: adenylosuccinate lyase
Note: GTP used to make
AMP, ATP used to make
GMP.
Also, feedback inhibition by
AMP and GMP.
51
Purine Nucleotide Synthesis
O
COO
OOC
2-
O3P O CH2
H
O
H

H
H
C
OH
OH
OH
Aspartate
+ ATP
CH
5
ADP
+ Pi
HC
N
H
SAICAR Synthetase
CH2
C
COO
Ribose
Phosphate
Pyrophosphokinase
AIR
Car boxylase
AMP
ADP + Pi
O3P O CH2
H

O
H
H
OH
OH
H
O
Ribose-5-Phosphate
Fumarate
P
O
O
O
P
O
C
O
C
H2N
CH
5
N
C
5-Aminoimidazole Ribotide (AIR)
ADP + Pi
N10-Formyl-
H2C
O3P O CH2
H
H
OH
OH
HN

O
C
H 2C
O
C
H
ADP +
Glutamate + Pi
FGAM
Synthetase
NH2
5-Formaminoimidazole-4-carboxamide
ribotide (FAICAR)
ATP +
Glutamine +
H2O
H 2C
IMP
Cyclohydrolase
O
C
NH
O
N10-Formyl-THF
THF
O
HN
C
O
HC
C5
NH
H
H
H
OH
Glycinamide Ribotide (GAR)
GAR Transformylase
N
CH
C
C
N
Ribose-5-Phosphate
H2O
H
N
CH
5
NH
Ribose-5-Phosphate
Formylglycinamidine ribotide (FGAM)
N
C4
NH
ADP
+ Pi
OH
C
H
GAR Synthetase
AICAR
Transformylase
THF
H2N
O
-5-Phosphoribosylamine (PRA)
Glycine
+ ATP
H
CH
C
NH2
O
H
O3P O CH2
THF
O
H
N
Glutamate
+ PPi
2-
5-Aminoimidazole-4-carboxamide
ribotide (AICAR)
ATP
Transferase
2-
N
Ribose-5-Phosphate
AIR
Synthetase
Glutamine
+ H2O
Amidophosphoribosyl
CH
5
H2N
Ribose-5-Phosphate
5-Phosphoribosyl--pyrophosphate (PRPP)
N
C4
H2N
O
Adenylosuccinate
Lyase
O
N
HC 4
N
5-Aminoimidazole-4-(N-succinylocarboxamide)
ribotide (SAICAR)
ATP
+HCO3
2-
CH
5
H2N
Ribose-5-Phosphate
Carboxyamidoimidazole Ribotide (CAIR)
N
C4
N
H2N
-D-Ribose-5-Phosphate (R5P)
ATP
C
N
C4
Ribose-5-Phosphate
Formylglycinamide ribotide (FGAR)
4
CH
N
N
2-
O3P O CH2
H
H
OH
O
H
H
OH
Inosine Monophosphate (IMP)
52
Nucleotide Metabolism
• PURINE RIBONUCLEOTIDES: formed de novo
– i.e., purines are not initially synthesized as free bases
– First purine derivative formed is Inosine Mono-phosphate
(IMP)
• The purine base is hypoxanthine
• AMP and GMP are formed from IMP
53
IMP Conversion to AMP
54
IMP Conversion to GMP
55
Regulatory Control of Purine Biosynthesis
• At level of IMP production:
– Independent control
– Synergistic control
– Feedforward activation by PRPP
• Below level of IMP production
– Reciprocal control
• Total amounts of purine nucleotides controlled
• Relative amounts of ATP, GTP controlled
56
Regulatory Control of Purine
Nucleotide Biosynthesis
• GTP is involved in AMP synthesis and ATP is involved in
GMP synthesis (reciprocal control of production)
• PRPP is a biosynthetically “central” molecule (why?)
– ADP/GDP levels – negative feedback on Ribose Phosphate
Pyrophosphokinase
– Amidophosphoribosyl transferase is activated by PRPP levels
– APRT activity has negative feedback at two sites
• ATP, ADP, AMP bound at one site
• GTP,GDP AND GMP bound at the other site
• Rate of AMP production increases with increasing
concentrations of GTP; rate of GMP production increases with
increasing concentrations of ATP
57
Pathways from AMP and GMP to ATP and GTP
Conversion to diphosphate involves specific kinases:
GMP + ATP <-------> GDP + ADP Guanylate kinase
AMP + ATP <-------> 2 ADP
Adenylate kinase
Conversion to triphosphate by Nucleoside diphosphate kinase (NDK):
GDP + ATP <------> GTP + ADP DG0’= 0
ping pong reaction mechanism with phospho-his intermediate.
NDK also works with pyrimidine nucleotides and is driven by mass action.
58
Allosteric regulation of purine de novo synthesis
59
Clinical disorders of purine metabolism
Excessive accumulation of uric acid: Gout
The three defects shown each result in elevated de novo
purine biosynthesis
60
Diseases of purine metabolism (continued)
Lesch-Nyhan Syndrome: Severe HGPRT deficiency
In addition to symptoms of gout, patients display severe behavioral
disorders, learning disorder, aggressiveness and hostility, including selfdirected. Patients must be restrained to prevent self-mutilation. Reason
for the behavioral disorder is unknown.
X-linked trait (HGPRT gene is on X chromosome).
Severe combined immune deficiency (SCID): lack of adenosine
deaminase (ADA).
Lack of ADA causes accumulation of deoxyadenosine.
Immune cells, which have potent salvage pathways, accumulate dATP,
which blocks production of other dNTPs by its action on ribonucleotide
reductase. Immune cells can’t replicate their DNA, and thus can’t
mount an immune response.
61
De novo pyrimidine biosynthesis
Pyrimidine ring is assembled as the free base, orotic acid, which is
them converted to the nucleotide orotidine monophosphate (OMP).
The pathway is unbranched. UTP is a substrate for formation of
CTP.
62
Pyrimidine Synthesis
O
2 ATP + HCO3- + Glutamine + H2O
C
2 ADP +
Glutamate +
Pi
O
Carbamoyl
Phosphate
Synthetase II
C
C
NH2
CH
C
N
H
PO3-2
O
PRPP
C
O
C
C
N
O
HN
O
CH
HN
PPi
2-
COO
O3P
O
Orotate Phosphoribosyl
Transferase
CH2
O
H
H
OH
OH
H

H
COO
Orotidine-5'-monophosphate
(OMP)
Orotate
Carbamoyl Phosphate
Aspartate
Reduced
Quinone
Aspartate
Transcarbamoylase
(ATCase)
O
O
O
C
C
O
CH
N
H
CH
O
2-
CH
N
H
COO
COO
O3P
O
CH2
CH
N
O
H2O
Dihydroorotase
Carbamoyl Aspartate
C
CH2
HN
C
C
C
HN
CH2
NH2
CO2
Quinone
Pi
HO
OMP
Decarboxylase
Dihydroorotate
Dehydrogenase
O
H
H
OH
OH
H

H
Dihydroorotate
Uridine Monophosphate
(UMP) 63
De novo synthesis of pyrimidines
1: carbamyl phosphate
synthase
2: aspartate
transcarbamylase
3: dihydroorotase
4: dihydroorotate DH
5: orotate
phosphoribosyl
tranferase
6: orotidylate
decarboxylase
7: UMP kinase
8: NDK
9: CTP synthetase
CAD=1,2,3
5 +6=single protein
64
UMP  UTP and CTP
• Nucleoside monophosphate kinase catalyzes
transfer of Pi to UMP to form UDP; nucleoside
diphosphate kinase catalyzes transfer of Pi from
ATP to UDP to form UTP
• CTP formed from UTP via CTP Synthetase
driven by ATP hydrolysis
– Glutamine provides amide nitrogen for C4
65
Regulation of
pyrimidine
de novo synthesis
66
Overview of dNTP biosynthesis
One enzyme, ribonucleotide reductase,
reduces all four ribonucleotides to their
deoxyribo derivitives.
A free radical mechanism is involved
in the ribonucleotide reductase
reaction.
There are three classes of ribonucleotide
reductase enzymes in nature:
Class I: tyrosine radical, uses NDP
Class II: adenosylcobalamin. uses NTPs
(cyanobacteria, some bacteria,
Euglena).
Class III: SAM and Fe-S to generate
radical, uses NTPs.
(anaerobes and fac. anaerobes).
67
Sources of reducing power for rNDP reductase
68
Thioredoxin
• Physiologic reducing
agent of RNR
• Cys pair can swap H atoms with disulfide formed
regenerate original enzyme
– Thioredoxin gets oxidized to disulfide
Oxidized Thioredoxin gets reduced by thioredoxin reductase mediated
by NADPH (final electron acceptor)
69
Relationship between thymidylate synthase and enzymes of
tetrahydrofolate metabolism
70
Tetrahydrofolate (THF)
• Methylation of dUMP catalyzed by thymidylate
synthase
– Cofactor: N5,N10-methylene THF
• Oxidized to dihydrofolate
• Only known rxn where net oxidation state of THF
changes
• THF Regeneration:
DHF + NADPH + H+  THF + NADP+ (enzyme: dihydrofolate reductase)
THF + Serine  N5,N10-methylene-THF + Glycine
(enzyme: serine hydroxymethyl transferase)
71
Thymine Formation
• Formed by methylating deoxyuridine
monophosphate (dUMP)
• UTP needed for RNA production, but dUTP not
needed for DNA
– If dUTP produced excessively, would cause substitution
errors (dUTP for dTTP)
• dUTP hydrolyzed by dUTP diphosphohydrolase to
dUMP  methylated at C5 to form dTMP
rephosphorylate to form dTTP
72
Salvage and de novo pathways to thymine nucleotides
73
Structure of rNDP reductase (E. coli, ClassI)
74
Proposed mechanism
for rNDP reductase
75
Proposed reaction mechanism for ribonucleotide reductase
76
Biological activities of thioredoxin
77
Regulation of activities of mammalian rNDP reductase
78
Substrate recvognition by dUTPase
79
Catalytic mechanism of thymidylate
synthase
80
Regeneration of N5, N10-methylenetetrahydrofolate
81
Biosynthesis of NAD+ and NADP+
82
Biosynthesis of CoA from pantothenate
83
Proposed reaction mechanism for FGAM synthetase
84
The transformylation reactions are catalyzed by a multiprotein complex
components of the complex:
GAR transformylase (3)
AICAR transformylase (9)
serine hydroxymethyl transferase, trifunctional formylmethenylmethylene-THF synthase (activities shown with asterisk)
85
Proposed catalytic mechanism for OMP decarboxylase
86
Reactions catalyzed by eukaryotic dihydroorotate dehydrogenase
87
Nitrogenous Bases
• Planar, aromatic, and heterocyclic
• Derived from purine or pyrimidine
• Numbering of bases is “unprimed”
88
Purine Nucleotide Synthesis
• ATP is involved in 6 steps and an additional ATP is needed to form
the first molecule (R5P)
• PRPP in the first step of Purine synthesis is also a precursor for
Pyrimidine Synthesis, His and Trp synthesis
– Role of ATP in first step is unique– group transfer rather than coupling
• In second step, C1 notation changes from  to  (anomers
specifying OH positioning on C1 with respect to C4 group)
• In step 3, PPi is hydrolyzed to 2Pi (irreversible, “committing” step)
89
Coupling of Reactions
• Hydrolyzing a phosphate from ATP is relatively easy
DG°’= -30.5 kJ/mol
– If endergonic reaction released energy into cell as heat energy,
wouldn’t be useful
– Must be coupled to an exergonic reaction
• When ATP is a reactant:
– Part of the ATP can be transferred to an acceptor: Pi, PPi, adenyl,
adenosinyl group in transferase reaction
OR
– ATP hydrolysis can drive an otherwise unfavorable reaction
(synthetase; “energase”)
or
90
Purine Biosynthetic Pathway
• Coupling of some reactions on pathway organizes and controls
processing of substrates to products in each step
– Increases overall rate of pathway and protects intermediates from
degradation
• In animals, IMP synthesis pathway is coupled:
– Reactions 3, 4, 6
– Reactions 7, 8
– Reactions 10, 11
91
92
Xanthosine Degradation
• Ribose sugar gets recycled (Ribose-1-Phosphate  R-5-P )
– can be incorporated into PRPP (efficiency)
• Hypoxanthine is converted to Xanthine by Xanthine Oxidase
• Guanine is converted to Xanthine by Guanine Deaminase
• Xanthine gets converted to Uric Acid by Xanthine Oxidase
93
Xanthine Oxidase
• A homodimeric protein
• Contains electron transfer proteins
– FAD
– Mo-pterin complex in +4 or +6 state
– Two 2Fe-2S clusters
• Transfers electrons to O2  H2O2
– H2O2 is toxic
– Disproportionated to H2O and O2 by catalase
94
THE PURINE NUCLEOTIDE CYCLE
AMP + H2O  IMP + NH4+
(AMP Deaminase)
IMP + Aspartate + GTP  AMP + Fumarate + GDP + Pi
(Adenylosuccinate Synthetase)
COMBINE THE TWO REACTIONS:
Aspartate + H2O + GTP  Fumarate + GDP + Pi + NH4+
The overall result of combining reactions is deamination of Aspartate to
Fumarate at the expense of a GTP
95
Purine Nucleotide Cycle
In-Class Question: Why is the purine nucleotide cycle
important in muscle metabolism during a burst of
activity?
96
Adenosine Deaminase
CHIME Exercise: 2ADA
• Enzyme catalyzing deamination of Adenosine to Inosine
• / barrel domain structure
– “TIM Barrel” – central barrel structure with 8 twisted
parallel -strands connected by 8
-helical loops
– Active site is at bottom of funnel-shaped pocket formed
by loops
– Found in all glycolytic enzymes
– Found in proteins that bind and transport metabolites
97
A CASE STUDY : GOUT
• A 45 YEAR OLD MAN AWOKE FROM SLEEP WITH A PAINFUL
AND SWOLLEN RIGHT GREAT TOE. ON THE PREVIOUS
NIGHT HE HAD EATEN A MEAL OF FRIED LIVER AND
ONIONS, AFTER WHICH HE MET WITH HIS POKER GROUP
AND DRANK A NUMBER OF BEERS.
• HE SAW HIS DOCTOR THAT MORNING, “GOUTY ARTHRITIS”
WAS DIAGNOSED, AND SOME TESTS WERE ORDERED. HIS
SERUM URIC ACID LEVEL WAS ELEVATED AT 8.0 mg/dL (NL <
7.0 mg/dL).
• THE MAN RECALLED THAT HIS FATHER AND HIS
GRANDFATHER, BOTH OF WHOM WERE ALCOHOLICS,
OFTEN COMPLAINED OF JOINT PAIN AND SWELLING IN
THEIR FEET.
98
A CASE STUDY : GOUT
• THE DOCTOR RECOMMENDED THAT THE MAN
USE NSAIDS FOR PAIN AND SWELLING, INCREASE
HIS FLUID INTAKE (BUT NOT WITH ALCOHOL)
AND REST AND ELEVATE HIS FOOT. HE ALSO
PRESCRIBED ALLOPURINOL.
• A FEW DAYS LATER THE CONDITION HAD
RESOLVED AND ALLOPURINOL HAD BEEN
STOPPED. A REPEAT URIC ACID LEVEL WAS
OBTAINED (7.1 mg/dL). THE DOCTOR GAVE THE
MAN SOME ADVICE REGARDING LIFE STYLE
CHANGES.
99
ALLOPURINOL IS A XANTHINE OXIDASE
INHIBITOR
A SUBSTRATE ANALOG IS CONVERTED TO AN
INHIBITOR, IN THIS CASE A “SUICIDE-INHIBITOR”
100
Lesch-Nyhan Syndrome
• A defect in production or activity of
HGPRT
– Causes increased level of Hypoxanthine and
Guanine ( in degradation to uric acid)
• Also,PRPP accumulates
– stimulates production of purine nucleotides (and
thereby increases their degradation)
• Causes gout-like symptoms, but also
neurological symptoms  spasticity,
aggressiveness, self-mutilation
• First neuropsychiatric abnormality that was
attributed to a single enzyme
101
Purine Autism
• 25% of autistic patients may
overproduce purines
• To diagnose, must test urine over 24
hours
– Biochemical findings from this test
disappear in adolescence
– Must obtain urine specimen in infancy,
but it’s difficult to do!
• Pink urine due to uric acid crystals may be
seen in diapers
102
Pyrimidine Ribonucleotide Synthesis
• Uridine Monophosphate (UMP) is
synthesized first
– CTP is synthesized from UMP
• Pyrimidine ring synthesis completed first;
then attached to ribose-5-phosphate
N1, C4, C5, C6 : Aspartate
C2 : HCO3N3 : Glutamine amide Nitrogen
103
UMP Synthesis Overview
• 2 ATPs needed: both used in first step
– One transfers phosphate, the other is hydrolyzed to ADP and Pi
• 2 condensation rxns: form carbamoyl aspartate and
dihydroorotate (intramolecular)
• Dihydroorotate dehydrogenase is an intra-mitochondrial
enzyme; oxidizing power comes from quinone reduction
• Attachment of base to ribose ring is catalyzed by OPRT;
PRPP provides ribose-5-P
– PPi splits off PRPP – irreversible
• Channeling: enzymes 1, 2, and 3 on same chain; 5 and 6 on
same chain
104
Pyrimidine Synthesis
O
2 ATP + HCO3- + Glutamine + H2O
C
2 ADP +
Glutamate +
Pi
O
Carbamoyl
Phosphate
Synthetase II
C
C
NH2
CH
C
N
H
PO3-2
O
PRPP
C
O
C
C
N
O
HN
O
CH
HN
PPi
2-
COO
O3P
O
Orotate Phosphoribosyl
Transferase
CH2
O
H
H
OH
OH
H

H
COO
Orotidine-5'-monophosphate
(OMP)
Orotate
Carbamoyl Phosphate
Aspartate
Reduced
Quinone
Aspartate
Transcarbamoylase
(ATCase)
O
O
O
C
C
O
CH
N
H
CH
O
2-
CH
N
H
COO
COO
O3P
O
CH2
CH
N
O
H2O
Dihydroorotase
Carbamoyl Aspartate
C
CH2
HN
C
C
C
HN
CH2
NH2
CO2
Quinone
Pi
HO
OMP
Decarboxylase
Dihydroorotate
Dehydrogenase
O
H
H
OH
OH
H

H
Dihydroorotate
Uridine Monophosphate
(UMP)105
106
OMP DECARBOXYLASE : THE MOST
CATALYTICALLY PROFICIENT ENZYME
• FINAL REACTION OF PYRIMIDINE PATHWAY
• ANOTHER MECHANISM FOR DECARBOXYLATION
• A CARBANION INTERMEDIATE (UNSTABLE)
– MUST BE STABILIZED
– BUT NO COFACTORS ARE NEEDED!
• SOME OF THE BINDING ENERGY BETWEEN OMP
AND THE ACTIVE SITE IS USED TO STABILIZE THE
TRANSITION STATE
– “PREFERENTIAL TRANSITION STATE BINDING”
107
108
Regulatory Control of Pyrimidine
Synthesis
• Differs between bacteria and animals
– Bacteria – regulation at ATCase rxn
• Animals – regulation at carbamoyl phosphate synthetase
II
– UDP and UTP inhibit enzyme; ATP and PRPP activate it
– UMP and CMP competitively inhibit OMP Decarboxylase
*Purine synthesis inhibited by ADP and GDP at ribose
phosphate pyrophosphokinase step, controlling level of
PRPP  also regulates pyrimidines
109
Orotic Aciduria
• Caused by defect in protein chain with enzyme
activities of last two steps of pyrimidine synthesis
• Increased excretion of orotic acid in urine
• Symptoms: retarded growth; severe anemia
• Only known inherited defect in this pathway
(all others would be lethal to fetus)
• Treat with uridine/cytidine
• IN-CLASS QUESTION: HOW DOES URIDINE AND
CYTIDINE ADMINISTRATION WORK TO TREAT
OROTICACIDURIA?
110
Degradation of Pyrimidines
• CMP and UMP degraded to bases similarly
to purines
– Dephosphorylation
– Deamination
– Glycosidic bond cleavage
• Uracil reduced in liver, forming -alanine
– Converted to malonyl-CoA  fatty acid
synthesis for energy metabolism
111
Deoxyribonucleotide Formation
• Purine/Pyrimidine degradation are the same for
ribonucleotides and deoxyribonucleotides
• Biosynthetic pathways are only for
ribonucleotides
• Deoxyribonucleotides are synthesized from
corresponding ribonucleotides
112
DNA vs. RNA: REVIEW
• DNA composed of deoxyribonucleotides
• Ribose sugar in DNA lacks hydroxyl group at 2’
Carbon
• Uracil doesn’t (normally) appear in DNA
– Thymine (5-methyluracil) appears instead
113
Formation of Deoxyribonucleotides
• Reduction of 2’ carbon done via a free radical
mechanism catalyzed by “Ribonucleotide
Reductases”
– E. coli RNR reduces ribonucleoside diphosphates
(NDPs) to deoxyribonucleoside diphosphates (dNDPs)
• Two subunits: R1 and R2
– A Heterotetramer: (R1)2 and (R2)2 in vitro
114
RIBONUCLEOTIDE REDUCTASE
• R1 SUBUNIT
–
–
–
–
Specificity Site
Hexamerization site
Activity Site
Five redox-active –SH groups from cysteines
• R2 SUBUNIT
– Tyr 122 radical
– Binuclear Fe(III) complex
115
Chime Exercise
E. coli Ribonucleotide Reductase:
3R1R and 4R1R: R1 subunit
1RIB and 1AV8: R2 subunit
116
Ribonucleotide Reductase R2
Subunit
• Fe prosthetic group– binuclear, with each Fe
octahedrally coordinated
– Fe’s are bridged by O-2 and carboxyl gp of Glu 115
– Tyr 122 is close to the Fe(III) complex  stabilization
of a tyrosyl free-radical
• During the overall process, a pair of –SH groups
provide the reducing equivalents
– A protein disulfide group is formed
– Gets reduced by two other sulfhydryl gps of Cys
residues in R1
117
Mechanism of Ribonucleotide Reductase
Reaction
• Free Radical
• Involvement of multiple –SH groups
• RR is left with a disulfide group that must
be reduced to return to the original enzyme
118
RIBONUCLEOTIDE REDUCTASE
• ACTIVITY IS RESPONSIVE TO LEVEL OF
CELLULAR NUCLEOTIDES:
– ATP ACTIVATES REDUCTION OF
• CDP
• UDP
– dTTP
• INDUCES GDP REDUCTION
• INHIBITS REDUCTION OF CDP. UDP
– dATP INHIBITS REDUCTION OF ALL NUCLEOTIDES
– dGTP
• STIMULATES ADP REDUCTION
• INHIBITS CDP,UDP,GDP REDUCTION
119
RIBONUCLEOTIDE REDUCTASE
• CATALYTIC ACTIVITY VARIES WITH STATE OF
OLIGOMERIZATION:
– WHEN ATP, dATP, dGTP, dTTP BIND TO SPECIFICITY SITE
OF R1 (CATALYTICALLY INACTIVE MONOMER)
•  CATALYTICALLY ACTIVE (R1)2
– WHEN dATP OR ATP BIND TO ACTIVITY SITE OF DIMERS
•  TETRAMER FORMATION
• (R1)4a (ACTIVE STATE) == (R1)4b (INACTIVE)
– WHEN ATP BINDS TO HEXAMERIZATION SITE
•  CATALYTICALLY ACTIVE HEXAMERS (R1)6
120
Anti-Folate Drugs
• Cancer cells consume dTMP quickly for DNA
replication
– Interfere with thymidylate synthase rxn to decrease
dTMP production
• (fluorodeoxyuridylate – irreversible inhibitor) – also affects
rapidly growing normal cells (hair follicles, bone marrow,
immune system, intestinal mucosa)
• Dihydrofolate reductase step can be stopped
competitively (DHF analogs)
– Anti-Folates: Aminopterin, methotrexate, trimethoprim
121
IN-CLASS QUESTION
• IN von GIERKE’S DISEASE, OVERPRODUCTION OF URIC ACID OCCURS. THIS
DISEASE IS CAUSED BY A DEFICIENCY OF
GLUCOSE-6-PHOSPHATASE.
– EXPLAIN THE BIOCHEMICAL EVENTS THAT
LEAD TO INCREASED URIC ACID
PRODUCTION?
– WHY DOES HYPOGLYCEMIA OCCUR IN THIS
DISEASE?
– WHY IS THE LIVER ENLARGED?
122
ADENOSINE DEAMINASE DEFICIENCY
• IN PURINE DEGRADATION, ADENOSINE 
INOSINE
– ENZYME IS ADA
• ADA DEFICIENCY RESULTS IN SCID
– “SEVERE COMBINED IMMUNODEFICIENCY”
• SELECTIVELY KILLS LYMPHOCYTES
– BOTH B- AND T-CELLS
– MEDIATE MUCH OF IMMUNE RESPONSE
• ALL KNOWN ADA MUTANTS STRUCTURALLY
PERTURB ACTIVE SITE
123