Nucleic Acids are the chemical carriers of a cell

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Nucleic Acids

• Nucleic Acids are the chemical carriers of a
  cells genetic information
• Deoxyribonucleic acid (DNA)
• Holds the information that determines the nature
  of a cell
• Controls cell growth and division
• Directs biosynthesis of the enzymes and other
  proteins required for cellular functions
• Ribonucleic acid (RNA)
• Nucleic acid derivatives such as ATP are involved
  as phosphorylating agents in many biochemical
  pathways
• Several important coenzymes, including NAD, FAD,
  and coenzyme A, have nucleic acid components

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24.1 Nucleotides and Nucleic Acids

• Nucleic acids are biopolymers
• Composed of nucleotides which are joined together
  to form a long chain
• Nucleotide
• Composed of nucleosides bound to a phosphate
  group
• Nucleoside
• Composed of an aldopentose sugar linked through
  its anomeric carbon to the nitrogen atom of a
  heterocyclic purine or pyrimidine base

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Nucleotides and Nucleic Acids

• DNA
• Sugar component is 2'-deoxyribose (the prefix
  2'-deoxy indicates that oxygen is missing from
  the 2' position of ribose)
• Contains four different amino bases
• Two substituted purines (adenine and guanine)
• Two substituted pyrimidines (cytosine and
  thymine)
• RNA
• Sugar component is ribose
• Contains adenine, guanine, and cytosine
• Thymine is replaced by a closely related
  pyrimidine base called uracil

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Nucleotides and Nucleic Acids

• The pyrimidines and purines found in DNA and RNA

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Nucleotides and Nucleic Acids

• Structures of the four deoxyribonucleotides

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Nucleotides and Nucleic Acids

• Structures of the four ribonucleotides

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Nucleotides and Nucleic Acids

• In naming and numbering nucleotides, positions on
  the sugars are given a prime superscript to
  distinguish them from positions on the amine base
• DNA and RNA differ dramatically in size
• Molecules of DNA have molecular weights up to 75
  billion
• Molecules of RNA are much smaller, containing as
  few as 21 nucleotides, and have a molecular
  weight as low as 7,000

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Nucleotides and Nucleic Acids

• Nucleotides are linked together in DNA and RNA by
  phosphodiester bonds between the phosphate group
  at C5' on one nucleotide and the 3'-hydroxyl
  group of the sugar of another nucleotide
• C3' is one free hydroxyl group at the end of the
  nucleic polymer (the 3' end)
• C5' is another free hydroxyl group at the other
  end of the nucleic polymer (the 5' end)
• Sequence of nucleotides in a chain is described
  by starting at the 5' end and identifying the
  bases in order of occurrence (using G, C, A, T or
  U)

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24.2 Base Pairing in DNA The Watson- Crick
Model

• Samples of DNA isolated from different tissues of
  the same species have the same proportions of
  heterocyclic bases
• Samples of DNA from different species often have
  greatly different proportions of bases
• Composition of human DNA
• 30 each of adenine and thymine
• 20 each of guanine and cytosine
• Composition of the bacterium Clostridium
  perfringens
• 37 each of adenine and thymine
• 13 each of guanine and cytosine

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Base Pairing in DNA The Watson-Crick Model

• In 1953, James Watson and Francis Crick proposed
  the secondary structure of DNA
• DNA under physiological conditions consists of
  two polynucleotide strands
• Strands run in opposite directions and coil
  around each other in a double helix
• The helix is 20 Å wide

• The two strands are complementary and are held
  together by hydrogen bonds between specific pairs
  of bases
• A with T
• C with G

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Base Pairing in DNA The Watson-Crick Model

• There are 10 base pairs per turn
• Each turn is 34 Å in length
• The two strands of the double helix coil in such
  a way that two kinds of grooves result
• A major groove 12 Å wide
• A minor groove 6 Å wide
• The grooves are lined with
  hydrogen
  bond donors and
  acceptors

• A variety of flat, polycyclic
  aromatic
  molecules are able
  to slip
  sideways, or intercalate,
  between the stacked
  bases
• An organisms genetic
  
  information is stored as a
  
  sequence of deoxyribonucleotides strung together
  in the DNA chain

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Base Pairing in DNA The Watson-Crick Model

• Central dogma of molecular genetics
• The function of DNA is to store information and
  pass it to RNA
• The function of RNA is to read, decode, and use
  the information received from DNA to make
  proteins
• Three fundamental processes take place
• Replication process by which identical copies
  of DNA are made so the information can be
  preserved and handed down to offspring
• Transcription the process by which the genetic
  messages are read and carried out of the cell
  nucleus to ribosomes, where protein synthesis
  occurs
• Translation the process by which the genetic
  messages are decoded and used to synthesize
  proteins

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Worked Example 24.1Predicting the
Complementary Base Sequence in Double-Stranded
DNA

• What sequence of bases on one strand of DNA is
  complementary to the sequence TATGCAT on another
  strand?

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Worked Example 24.1Predicting the
Complementary Base Sequence in Double-Stranded
DNA

• Strategy
• Remember that A and G form complementary pairs
  with T and C
• Go through the sequence replacing A by T, G by C,
  T by A, and C by G
• Remember that the 5' end is on the left and the
  3' end is on the right in the original strand

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Worked Example 24.1Predicting the
Complementary Base Sequence in Double-Stranded
DNA

• Solution
• Original (5') TATGCAT (3')
• Compliment (3') ATACGTA (5') or
• (5') ATGCATA (3')

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24.3 Replication of DNA

• Replication
• An enzyme-catalyzed process
• Begins with a partial unwinding of the double
  helix
• Bases become exposed
• New nucleotides line up on each strand in a
  complementary manner (A with T and C with G)
• Two new strands begin to grow
• Each new strand is complementary to its old
  template strand
• Two identical DNA helices are produced
• The process is described as semiconservative
  replication because each of the new DNA molecules
  contains one old strand and one new strand

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Replication of DNA

• A representation of semiconservative DNA
  replication

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Replication of DNA

• Addition of nucleotides to the growing chain
• Takes place in the 5'?3' direction
• Catalyzed by DNA polymerase
• Key step is the addition of a nucleoside
  5'-triphosphate to the free 3'-hydroxyl group of
  the growing chain, with loss of a diphosphate
  leaving group

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Replication of DNA

• Both new strands are synthesized in the 5'?3'
  direction
• They cannot be made in exactly the same way
• One strand must have its 3' end near the point of
  unraveling (the replication fork), while the
  other strand has its 5' end near the replication
  fork
• The complement of the original 5'?3' strand is
  synthesized continuously in a single piece
• The compliment of the original 3'?5' strand is
  synthesized discontinuously in small pieces that
  are often then linked by DNA ligases

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24.4 Transcription of DNA

• RNA
• Similar to DNA but contains ribose instead of
  deoxyribose and uracil instead of thymine
• Three primary kinds
• Messenger RNA (mRNA) carries genetic messages
  from DNA to ribosomes,
• Small granular particles in the cytoplasm of a
  cell where protein synthesis takes place
• Ribosomal RNA (rRNA) complexed with protein
  provides the physical makeup of the ribosomes
• Transfer RNA (tRNA) transports amino acids to the
  ribosomes where they are joined together to make
  proteins

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Transcription of DNA

• Genetic information in DNA is contained in
  segments called genes
• Each gene consists of a specific nucleotide
  sequence that encodes a specific protein
• Conversion of DNA information into proteins
  begins with transcription of DNA to mRNA

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Transcription of DNA

• Promoter site
• A specific base sequence found within a DNA chain
  typically consisting of around 40 base pairs
  located upstream (5') of the transcription start
  site
• Consists of two hexameric consensus sequences,
  one located 10 base pairs upstream and the second
  located 35 base pairs upstream from the beginning
  of the coding region
• Signals the beginning of a gene
• Other base sequences signal a stop near the end
  of the gene

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Transcription of DNA

• Transcription
• The process by which genetic information encoded
  in DNA is read and used to synthesize RNA in the
  nucleus of the cell
• Several turns of the DNA double helix unwind,
  forming a bubble and exposing the bases of the
  two strands
• Ribonucleotides line up in the proper order by
  hydrogen bonding to their complementary bases on
  DNA
• Bond formation occurs in the 5' 3' direction
• The growing RNA molecule unwinds from DNA

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Transcription of DNA

• Only one of the two DNA strands is transcribed
  into mRNA
• The strand that contains the gene is called the
  coding strand, or sense strand
• The strand that gets transcribed is called the
  template strand, or antisense strand
• The RNA molecule produced during transcription is
  the complement of the DNA antisense strand and is
  therefore a copy of the DNA coding strand (except
  T has been replaced with U)

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Transcription of DNA

• Genes are not continuous segments of the DNA
  chain
• A gene begins in an exon, a small section of DNA
• Genes are interrupted by noncoding sections
  called introns
• Genes take up again further down the chain in
  another exon
• The final mRNA molecule results after the
  noncoded sections are cut out and the remaining
  pieces are spliced together
• 90 of human DNA seems to be made up of introns
• 10 of DNA contains coding instructions

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24.5 Translation of RNA Protein Biosynthesis

• Primary cellular function of mRNA
• Direct biosynthesis of the thousands of diverse
  peptides and proteins required by an organism
• The mechanics of protein biosynthesis take place
  on ribosomes, small granular particles in the
  cytoplasm of a cell that consist of about 60
  ribosomal RNA and 40 protein
• The specific ribonucleotide sequence in mRNA
  forms a codon that determines the order in which
  amino acid residues are joined
• Each codon consists of a sequence of three
  ribonucleotides that is specific for a given
  amino acid
• The series UUC on mRNA is a codon directing
  incorporation of the amino acid phenylalanine
  into the growing protein
• 64 possible triplets of the four bases in RNA
• 61 code for specific amino acids
• 3 code for chain termination

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Translation of RNA Protein Biosynthesis
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Translation of RNA Protein Biosynthesis

• Translation
• The process by which the genetic information
  transcribed from DNA onto mRNA is read by tRNA
  and used to direct protein synthesis
• There are 61 different tRNAs, one for each of the
  61 codons that specifies an amino acid
• A typical tRNA is single-stranded and
  cloverleaf-shaped
• On the middle leaf it contains an anticodon, a
  sequence of three ribonucleotides complementary
  to the codon sequence
• Contains about 70 to 100 ribonucleotides
• Bonded to a specific amino acid by an ester
  linkage through the 3' hydroxyl on ribose at the
  3' end of the tRNA

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Translation of RNA Protein Biosynthesis

• The codon sequence UUC present on mRNA is read by
  a phenylalanine-bearing tRNA having the
  complementary anticodon base sequence GAA
• Nucleotide sequences are written in the 5'?3'
  direction so the sequence in an anticodon must be
  reversed

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Translation of RNA Protein Biosynthesis

1. Successive codons on mRNA are read
2. Different tRNAs bring the correct amino acids
   into position for enzyme-mediated transfer to the
   growing peptide
3. When synthesis of the proper protein is
   completed, a stop codon signals the end
4. The protein is released from the ribosome

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Worked Example 24.2Predicting the Amino Acid
Sequence Transcribed from DNA

• What amino acid sequence is coded by the
  following segment of a DNA coding strand?
• (5') CTA-ACT-AGC-GGG-TCG-CCG (3')

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Worked Example 24.2Predicting the Amino Acid
Sequence Transcribed from DNA

• Strategy
• The mRNA produced during translation is a copy of
  the DNA coding strand
• Each T replaced by U
• The mRNA has the sequence
• (5') CUA-ACU-AGC-GGG-UCG-CCG (3')

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Worked Example 24.2Predicting the Amino Acid
Sequence Transcribed from DNA

• Solution
• Leu-Thr-Ser-Gly-Ser-Pro

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24.6 DNA Sequencing

• Methods for sequencing immense DNA chains
• First step in sequencing
• Cleave the DNA chain at known points to produce
  smaller pieces, done through the use of
  restriction endonucleases
• More than 3500 restriction enzymes are known
• About 200 restriction enzymes are commercially
  available
• Each different restriction enzyme cleaves a DNA
  molecule at a point in the chain where a specific
  base sequence occurs
• The restriction enzyme AluI cleaves between G and
  C in the four-base sequence AG-CT
• (5'-AGCT-(3') sequence is that same as its
  complement (3')-TCGA-(5') when both are read in
  the same 5'?3' direction

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DNA Sequencing

• Two methods of DNA sequencing are available
• The Maxam-Gilbert method
• Uses chemical techniques
• Sanger dideoxy method
• Uses enzymatic reactions
• The more commonly used of the two
• Method responsible for sequencing the entire
  human genome of 2.9 billion base pairs
• In commercial sequencing instruments, the dideoxy
  method begins with a mixture of the following
• The restriction fragment to be sequenced

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DNA Sequencing

• A small piece of DNA called a primer, whose
  sequence is complementary to that on the 3' end
  of the restriction fragment
• The four 2'-deoxyribonucleoside triphosphates
  (dNTPs)
• Very small amounts of the four 2'
  ,3'-dideoxyribonucleoside triphosphates (ddNTPs),
  each of which is labeled with a fluorescent dye
  of a different color
• (A 2' ,3' -dideoxyribonucleoside triphosphate in
  one in which both 2' and 3' OH groups are
  missing from ribose)

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DNA Sequencing

• DNA polymerase is added to the mixture
• A strand of DNA complementary to the restriction
  fragment begins to grow from the end of the
  primer
• Most of the time only normal deoxyribonucleotides
  are incorporated into the growing chain
• Sometimes a dideoxyribonucleotide is incorporated
• When this occurs, DNA synthesis stops because the
  chain end no longer has a 3' hydroxyl group for
  adding further nucleotides
• The product
• Consists of a mixture of DNA fragments of all
  possible lengths, each terminated by one of the
  four dye-labeled dideoxyribonucleotides
• Mixture is then separated according to the size
  of the pieces by gel electrophoresis

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DNA Sequencing

• The identity of the terminal dideoxyribonucleotide
  in each piece and thus the sequence of the
  restriction fragment is identified by noting
  the color with which it fluoresces

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24.7 DNA Synthesis

• Synthesis of short DNA segments, called
  oligonucleotides or oligos
• A nucleotide has multiple reactive sites that
  must be selectively protected and deprotected at
  the proper times
• Coupling of the four nucleotides must be carried
  out in the proper sequence
• Automated DNA synthesizers allow the fast and
  reliable synthesis of DNA segments up to 200
  nucleotides in length
• A protected nucleotide is covalently bonded to a
  solid support
• One nucleotide at a time is added to the growing
  chain by the use of a coupling reagent
• After the final nucleotide has been added, all
  the protecting groups are removed and the
  synthetic DNA is cleaved from the solid support

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DNA Synthesis

• Step 1 Attachment of a protected deoxynucleoside
  to a silica (SiO2) support
• Done through an ester linkage to the 3' OH
  group of the deoxynucleoside
• Both the 5' OH group on the sugar and free NH2
  groups on the heterocyclic bases must be
  protected
• The deoxyribose 5' OH is protected as its
  p-dimethoxytrityl (DMT) ether

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DNA Synthesis

• Adenine and cytosine bases are protected by
  benzoyl groups
• Guanine is protected by an isobutryl group
• Thymine requires no protection

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DNA Synthesis

• Step 2 Removal of the DMT protecting group by
  treatment with dichloroacetic acid in CH2Cl2
• Reaction occurs by an SN1 mechanism
• Reaction proceeds rapidly due to the stability of
  the tertiary, benzylic dimethoxytrityl cation

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DNA Synthesis

• Step 3 Coupling of the polymer-bonded
  deoxynucleoside with a protected deoxynucleoside
  containing a phosphoramidite group, R2NP(OR)2, at
  the 3' position
• Takes place in the polar aprotic solvent
  acetonitrile
• Requires catalysis by the heterocyclic amine
  tetrazole
• Yields a phosphite, P(OR)3

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DNA Synthesis

• Step 4 Oxidation
• Phosphite product is oxidized to a phosphate by
  treatment with iodine in aqueous tetrahydrofuran
  in the presence of 2,6-dimethylpyridine
• The cycle is repeated until oligonucleotide chain
  of the desired sequence is built
• Deprotection
• Coupling
• Oxidation

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DNA Synthesis

• Step 5 Final step
• Removal of all protecting groups
• Cleavage of the ester bond holding the DNA to the
  silica
• All reactions are done at the same time by
  treatment with aqueous NH3
• Purification by electrophoresis yields the
  synthetic DNA

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24.8 The Polymerase Chain Reaction

• Polymerase chain reaction (PCR)
• A method for amplifying small amounts of DNA to
  produce larger amounts
• Invented by Kary Mullis in 1986
• PCR produces multiple copies of a given DNA
  sequence
• Makes it possible to obtain several micrograms (1
  ug 10-6 g about 1011 nucleotides) in a few
  hours when starting from less than 1 picogram of
  DNA with a chain length of 10,000 nucleotides
  (1 pg 10-12 g about 100,000 molecules)

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The Polymerase Chain Reaction

• Taq polymerase
• The key to the polymerase chain reaction
• A heat-stable enzyme isolated from the
  thermophilic bacterium Thermus aquaticus found in
  a hot spring in Yellowstone National Park
• Able to take a single strand of DNA that has a
  short, primer segment of complementary chain at
  one end and then finish constructing the entire
  complementary strand
• Overall process takes three steps

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The Polymerase Chain Reaction

• The polymerase chain reaction

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The Polymerase Chain Reaction

• Step 1 Denaturation of the double-stranded DNA
• The double-stranded DNA is heated in the presence
  of
• Taq polymerase
• Mg2 ion
• The four deoxynucleotide triphosphate monomers
  (dNTPs)
• A large excess of two short oligonucleotide
  primers of about 20 bases each
• Each primer is complementary to the sequence at
  the end of one of the target DNA segments
• Double-stranded DNA denatures at a temperature of
  95 ºC, spontaneously breaking apart into two
  single strands

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The Polymerase Chain Reaction

• Step 2
• The temperature is lowered
• Between 37 and 50 ºC
• Allows primers to anneal by hydrogen bonding to
  their complementary sequence at the end of each
  target strand
• Step 3
• Temperature is raised to 72 ºC
• Taq polymerase catalyzes the addition of further
  nucleotides to the two primed DNA strands
• When replication is finished, two copies of the
  original DNA exist
• Automated PCR
• 30 or so cycles can be carried out in an hour
  resulting in a theoretical amplification factor
  of 230 (109)
• The efficiency of each cycle is less than 100
• Experimental amplification is about 106 to 108

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24.9 Catabolism of Nucleotides

• Dietary nucleic acids
• First pass through the stomach to the intestines
• Hydrolyzed to their constituent nucleotides by a
  variety of different nucleases
• Dephosphorylation by various nucleotidases gives
  nucleosides
• A third cleavage by nucleosidases gives the
  constituent bases
• Bases are catabolized to produce intermediates
  that enter other metabolic processes

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Catabolism of Nucleotides

• Purine Catabolism Guanosine
• Guanosine and deoxyguanosine are both catalyzed
  by a three-step pathway
• Begins with cleavage to give guanine
• Guanine is hydrolyzed to yield xanthine
• Oxidation of xanthine gives uric acid, which is
  excreted in the urine

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Catabolism of Nucleotides

• Figure 24.10
• Pathway for the catabolism of guanosine and
  deoxyguanosine to uric acid

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Catabolism of Nucleotides

• STEP 1 OF FIGURE 24.10 PHOSPHOROLYSIS
• Phosphorolysis of guanosine (or deoxyguanosine)
• Catalyzed by purine nucleoside phosphorylase
• Gives b-ribose 1-phosphate (or b-deoxyribose
  1-phosphate) plus guanine
• Reaction probably occurs by an SN1-like
  replacement of guanine by phosphate ion through
  an oxonium-ion intermediate

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Catabolism of Nucleotides

• STEP 2 OF FIGURE 24.10 HYDROLYSIS
• Hydrolysis of guanine
• Gives xanthine
• Catalyzed by guanine deaminase
• Occurs by nucleophilic addition of water to the
  CN bond followed by expulsion of ammonium ion

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Catabolism of Nucleotides

• STEP 3 OF FIGURE 24.10 OXIDATION
• Xanthine
• Oxidized by xanthine oxidase
• A complex enzyme that contains FAD and an
  oxo-molybdenum(VI) cofactor
• A base deprotonates the Mo-OH group
• The resulting anion does a nucleophilic addition
  to the CN bond in xanthine
• The nitrogen anion expels hydride ion
• Hydride ion adds to an MoS bond, thereby
  reducing the molybdenum center from Mo(VI) to
  Mo(IV)
• Hydrolysis of the Mo-O bond gives an enol
• Enol tautomerizes to uric acid
• The reduced molybdenum is reoxidized by O2 in a
  complex redox pathway

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Catabolism of Nucleotides

• The mechanism of step 3 in Figure 24.10,
  oxidation of xanthine to yield uric acid

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Catabolism of Nucleotides

• Adenosine (purine nucleotide)
• Steps are similar to those for guanosine but
  order of steps differs
• Base in adenosine is first degraded and then
  removed

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24.10 Biosynthesis of Nucleotides

• Purine Biosynthesis Adenosine Monophosphate and
  Guanosine Monophosphate
• Purine nucleotides are formed by the initial
  attachment of an NH2 group to the ribose,
  followed by multistep buildup of the heterocyclic
  base
• -NH2 attaches by a nucleophilic substitution
  reaction of ammonia
• Inosine monophosphate (IMP) is the first fully
  formed purine ribonucleotide
• Adenosine monophosphate (AMP) derived from IMP

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Biosynthesis of Nucleotides

• Adenosine monophosphate
• Biosynthesized from IMP
• AMP is biosynthesized in a three-step sequence
• Initial phosphorylation with GTP to form an imino
  phosphate
• Nucleophilic acyl substitution reaction
• Reaction with aspartate to give adenylosuccinate
• Elimination of fumarate
• E1cB reaction

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Biosynthesis of Nucleotides

• Pathway for the conversion of inosine
  monophosphate to adenosine monophosphate