Non protein nitrogen compounds metabolism

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Non protein nitrogen compounds metabolism

• Porphyrins Nuleobases
  

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Heme Metabolism

• Heme biosynthesis
• Heme degradation

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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)

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• Two ALA molecules are joined in the presence of
  the enzyme delta aminolevulinic dehydratase
• Forms porphobilinogen
• Lead inhibits this step

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• Four porphobilinogen molecules condense to form
  hydroxymethylbilane and then uroporphyrinogen III
• Requires porphobilinogen deaminase
  (uroporphyrinogen synthtase) and uroporphyrinogen
  III co-synthtase

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• 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

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• Conversion of coproporphyrinogen III to
  protoporphyrinogen III
• Two propionic acid (CH2-CH2-COOH) convert to two
  vinyl (CH2CH2)
• Requires coproporphyrinogen oxidase and oxygen as
  a hydrogen acceptor
• Moves heme synthesis back into the mitochondria

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• 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

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Heme

• A complex of iron and protoporphyrin (a
  porphyrin ring)

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Porphyrins

• Protoporphyrin
• Coproporphyrin
• Uroporphyrin

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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.
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Heme Biosynthesis Porphyrias

• Cruelly referred to as a Vampires disease.
• Can be caused by lead poisoning The fall of the
  Roman Empire!

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Not a vampires 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 reasonsPorphyria is a rare,
but frightening condition hard to diagnose and
there is no cure.
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PORPHYRIAS
Mitochondria
GLYCINE SuccinylCoA
Agent Orange
ALA synthase
3p21/Xp11.21
d-aminolevulinic acid(ALA)
ALA-dehydratase Deficiency porphyria
ALA dehydratase
9q34
Porphobilinogen(PBG)
Acute intermittent porphyria
PBG deaminase
11q23
hydroxymethylbilane
Congenital erythropoietic porphyria
Uroporphyrinogen III cosynthase
10q26
uroporphyrinogen III
Uroporphyrinogen decarboxylase
Prophyria cutanea tarda
1q34
coprophyrinogene III
Coproporphyrinogen oxidase
Herediatary coproporphyria
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Protoporphyrinogene IX
Protoporphyrinogen oxidase
Variegate porphyria
protoporphyrin IX
1q14
Ferrochelatase
Erythropoietic protoporphyria
Heme
18q21.3
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porphyrias
Type Enzyme Involved Major Symptoms Laboratory tests
Acute intermittent porphyria Uroporphyrinogen synthase Abdominal painNeuropsychiatric urinary porphobilinogen ?
Congenital erythropoietic porphyria Uroporphyrinogen cosynthase Photosensitivity urinary uroporphyrin ? porphobilinogen ?
Porphyria cutanea tarda Decarboxylase Photosensitivity urinary uroporphyrin ? porphobilinogen ?
Variegate porphyria Oxidase PhotosensitivityAbdominal painNeuropsychiatric urinary uroporphyrin ? fecal coproporphyrin ? fecal protoporphyrin ?
Erythropoietic protoporphyria Ferrochelatase Photosensitivity fecal protoporphyrin ? red cell protoporphyrin ?
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Heme Catabolism
Heme Degradation
HEME
O2
(opens the porphyrin ring)
Fe3
BILIVERDIN
BILIRUBIN
BILIRUBIN diglucuronide
BILE
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BLOOD CELLS
KIDNEY
reabsorbed into blood
INTESTINE
via bile duct to intestines
LIVER
Figure 2. Catabolism of hemoglobin
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Jaundice
Hyperbilirubinemia Two forms Direct bilirubin
Conjugated with glucoronic acid Indirect
bilirubin unconjugated, insoluble in water.
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Whats 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.

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• 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
• bExtrahepatic cholestasis
• Blockage of bilirubin transport in the bilary
  tract.
• Increased direct bilirubin.
• Stool color turns gray.
• Caused by Tumors or gall stones.

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Examples of hyperbilirubinemia
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Genetic Disorders of Bilirubin Metabolism
Condition Defect Bilirubin Clinical Findings
Crigler-Najjar syndrome severely defective UDP-glucuronyltransferase Unconjugated bilirubin ??? Profound jaundice
Gilberts syndrome reduced activity of UDP-glucuronyltransferase Unconjugated bilirubin ? Very mild jaundice during illnesses
Dubin-Johnson syndrome abnormal transport of conjugated bilirubin into the biliary system Conjugated bilirubin ?? Moderate jaundice
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Nucleotides Synthesis and Degradation
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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).

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Nitrogenous Bases
Purines
Pyrimidines
N1 Aspartate Amine C2, C8 Formate N3, N9
Glutamine C4, C5, N7 Glycine C6 Bicarbonate Ion
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Purine degredation
AMP deamination in muscle, hydrolysis in other
tissues. Xanthine oxidasecontains FAD,
molybdenum, and non-heme iron. In primates, uric
acid is the end product, which is excreted.
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Purine Nucleotides

• Get broken down into Uric Acid (a purine)

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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.
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Purine degredation in other animals
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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

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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

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Catabolism of pyrimidines
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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.
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Reutilization and catabolism of purine and
pyrimidine bases
blue-catabolism red-salvage pathways
endonucleases pancreatic RNAse pancreatic
DNAse phosphodiesterases usually non-specific
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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.

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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)

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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

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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
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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.
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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 !

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De novo biosynthesis of purines low molecular
weight precursors of the purine ring atoms
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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.
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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.
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Purine Nucleotide Synthesis
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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

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IMP Conversion to AMP
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IMP Conversion to GMP
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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

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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

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Pathways from AMP and GMP to ATP and GTP
Conversion to diphosphate involves specific
kinases GMP ATP lt-------gt GDP ADP
Guanylate kinase AMP ATP lt-------gt 2
ADP Adenylate kinase Conversion to triphosphate
by Nucleoside diphosphate kinase (NDK) GDP
ATP lt------gt GTP ADP DG0 0 ping pong
reaction mechanism with phospho-his
intermediate. NDK also works with pyrimidine
nucleotides and is driven by mass action.
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Allosteric regulation of purine de novo synthesis
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Clinical disorders of purine metabolism
Excessive accumulation of uric acid Gout
The three defects shown each result in elevated
de novo purine biosynthesis
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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
self-directed. 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 cant replicate their DNA, and thus cant
mount an immune response.
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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.
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Pyrimidine Synthesis
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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 CAD1,2,3 5
6single protein
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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

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Regulation of pyrimidine de novo synthesis
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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). Cl
ass III SAM and Fe-S to generate radical, uses
NTPs. (anaerobes and fac. anaerobes).
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Sources of reducing power for rNDP reductase
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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)
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Relationship between thymidylate synthase and
enzymes of tetrahydrofolate metabolism
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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)

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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

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Salvage and de novo pathways to thymine
nucleotides
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Structure of rNDP reductase (E. coli, ClassI)
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Proposed mechanism for rNDP reductase
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Proposed reaction mechanism for ribonucleotide
reductase
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Biological activities of thioredoxin
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Regulation of activities of mammalian rNDP
reductase
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Substrate recvognition by dUTPase
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Catalytic mechanism of thymidylate synthase
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Regeneration of N5, N10-methylenetetrahydrofolate
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Biosynthesis of NAD and NADP
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Biosynthesis of CoA from pantothenate
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Proposed reaction mechanism for FGAM synthetase
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The transformylation reactions are catalyzed by a
multiprotein complex
components of the complex GAR transformylase
(3) AICAR transformylase (9) serine hydroxymethyl
transferase, trifunctional formylmethenyl-methylen
e-THF synthase (activities shown with asterisk)
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Proposed catalytic mechanism for OMP decarboxylase
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Reactions catalyzed by eukaryotic dihydroorotate
dehydrogenase
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Nitrogenous Bases

• Planar, aromatic, and heterocyclic
• Derived from purine or pyrimidine
• Numbering of bases is unprimed

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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 a to b
  (anomers specifying OH positioning on C1 with
  respect to C4 group)
• In step 3, PPi is hydrolyzed to 2Pi
  (irreversible, committing step)

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Coupling of Reactions

• Hydrolyzing a phosphate from ATP is relatively
  easy
• ?G -30.5 kJ/mol
• If endergonic reaction released energy into cell
  as heat energy, wouldnt 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, or adenosinyl group
  in transferase reaction
• OR
• ATP hydrolysis can drive an otherwise unfavorable
  reaction
• (synthetase energase)

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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

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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

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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

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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

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Purine Nucleotide Cycle

• In-Class Question Why is the purine nucleotide
  cycle important in muscle metabolism during a
  burst of activity?

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Adenosine Deaminase

• CHIME Exercise 2ADA
• Enzyme catalyzing deamination of Adenosine to
  Inosine
• a/b barrel domain structure
• TIM Barrel central barrel structure with 8
  twisted parallel b-strands connected by 8
  a-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

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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 lt 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.

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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.

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ALLOPURINOL IS A XANTHINE OXIDASE INHIBITORA
SUBSTRATE ANALOG IS CONVERTED TO AN INHIBITOR, IN
THIS CASE A SUICIDE-INHIBITOR
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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

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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 its
  difficult to do!
• Pink urine due to uric acid crystals may be seen
  in diapers

103
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 HCO3- N3
Glutamine amide Nitrogen
104
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

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Pyrimidine Synthesis
106


107
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

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109
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

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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?

111
Degradation of Pyrimidines

• CMP and UMP degraded to bases similarly to
  purines
• Dephosphorylation
• Deamination
• Glycosidic bond cleavage
• Uracil reduced in liver, forming b-alanine
• Converted to malonyl-CoA ? fatty acid synthesis
  for energy metabolism

112
Deoxyribonucleotide Formation

• Purine/Pyrimidine degradation are the same for
  ribonucleotides and deoxyribonucleotides
• Biosynthetic pathways are only for
  ribonucleotides
• Deoxyribonucleotides are synthesized from
  corresponding ribonucleotides

113
DNA vs. RNA REVIEW

• DNA composed of deoxyribonucleotides
• Ribose sugar in DNA lacks hydroxyl group at 2
  Carbon
• Uracil doesnt (normally) appear in DNA
• Thymine (5-methyluracil) appears instead

114
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

115
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

116
Chime Exercise

• E. coli Ribonucleotide Reductase
• 3R1R and 4R1R R1 subunit
• 1RIB and 1AV8 R2 subunit

117
Ribonucleotide Reductase R2 Subunit

• Fe prosthetic group binuclear, with each Fe
  octahedrally coordinated
• Fes 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

118
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

119
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

120
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

121
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

122
IN-CLASS QUESTION

• IN von GIERKES DISEASE, OVERPRO- DUCTION 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?

123
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