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1
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King Saud University College of
Science Department of Biochemistry

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Part 3 Coenzymes-Dependent Enzyme
MechanismProfessor A. S. Alhomida
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• Mechanism of Carbanion Stabilization by PLP

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Mechanism of Carbanion Stabilization by PLP
a
b
Internal aldimine (PLP-Enz Schiff base)
External aldimine (PLP-substrate
Schiff base)
a-Amino Acid
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Mechanism of Carbanion Stabilization by PLP,
Contd
a
b
b
a
Stabilized carbanion resonance
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Mechanism of Carbanion Stabilization by PLP,
Contd
a
b
a
b
Stabilized carbanion resonance
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Mechanism of Carbanion Stabilization by PLP,
Contd
a
b
a
b
Stabilized carbanion resonance
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Mechanism of Carbanion Stabilization by PLP,
Contd
a
a
b
b
Stabilized carbanion resonance
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Mechanism of Carbanion Stabilization by PLP,
Contd
a
a
b
b
Stabilized carbanion resonance
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Mechanism of Carbanion Stabilization by PLP,
Contd
b
a
a
b
For determination of stereochemistry of amino
acid formed
Stabilized carbanion resonance
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Mechanism of Carbanion Stabilization by PLP,
Contd
b
b
a
a
Stabilized carbanion resonance
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Jencks Statement

• The versatile chemistry of pyridoxal phosphate
  offers a rich learning experience for the student
  of mechanistic chemistry
• Professor W. Jencks, in his classic text,
  Catalysis in Chemistry and Enzymology, writes
• It has been said that God created an organism
  especially adapted to help the biologist find an
  answer to every question about the physiology of
  living systems

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Jencks Statement, Contd

• if this is so it must be concluded that pyridoxal
  phosphate was created to provide satisfaction and
  enlightenment to those enzymologists and chemists
  who enjoy pushing electrons, for no other
  coenzyme is involved in such a wide variety of
  reactions, in both enzyme and model systems,
  which can be reasonably interpreted in terms of
  the chemical properties of the coenzyme

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Jencks Statement, Contd

• Most of these reactions are made possible by a
  common structural feature
• That is, electron withdrawal toward the cationic
  nitrogen atom of the imine and into the electron
  sink of the pyridoxal ring from the a carbon atom
  of the attached amino acid activates all three of
  the substituents on this carbon atom for
  reactions which require electron withdrawal from
  this atom
• Jencks, William P., 1969. Catalysis in Chemistry
  and Enzymology. New York McGraw-Hill

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Biochemical Functions of Pyridoxal phosphate

• Decarboxylation of amino acids
• Transaminase reactions
• Racemization reactions
• Aldol cleavage reactions
• Transulfuration reactions
• Conversion of tryptophan to niacin
• Conversion of linoleic acid into arachidonic acid
  (prostaglandin precursor)
• Formation of sphingolipids

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• Transamination Reactions

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• The term transamination refers to the
  interconversion of carbonyl and amino groups.
  Condensation of an amine with an aldehyde as
  shown below gives an imine. What is formally only
  a tautomerisation reaction converts imine A into
  its tautomer B which upon hydrolysis yields the
  "transaminated" products. i.e. the product in
  which the amine and the carbonyl group have been
  swapped..

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• This is a highly simplified view of the
  transamination reaction.
• Firstly, aldehydes do not occur in biological
  systems due to their chemical instability. The
  biological equivalent of aldehydes are imines.

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• Firstly, aldehydes do not occur in biological
  systems due to their chemical instability. The
  biological equivalent of aldehydes are imines.

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• Secondly, imines are chemically stable towards
  this type of tautomerisation reaction. An enzyme
  is required to effect this transformation and the
  enzymes employs a co-factor (or prosthetic
  group).This cofactor is pyridoxal phosphate
  (PLP).

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• PLP is attached to the enzyme forms an imine with
  a lysine residue. This link attaches the
  co-factor to the enzyme and converts the aldehyde
  into its biological equivalent, the imine.
• The conversion of amino acids into a-keto acids
  (also sometimes referred to as a-oxo-acids) is a
  central reaction of primary and secondary
  metabolism.

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• In the first step of transamination reactions,
  pyridoxalphosphate in its biological form of
  imine is tranferred to the substrate amino acid.

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• Then the PLP-dependent enzymes catalyses the
  tautomerisation of the imime.

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• In the final step, hydrolysis of the imine gives
  the products.
• Note, that pyridoxal phosphate (PLP) has been
  converted into pyridoxamine by the transamination
  reaction. A second transamination step is
  required to convert pyridoxamine back into PLP.
  This restores the co-factor and the enzyme can
  carry out another transamination reaction.

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Mechanism of PLP-catalysed transaminations

• The a-hydrogen of the imine is in conjugation
  with the protonated pyridinium nitrogen. The
  positively chareged nitrogen increases the
  aciditiy of the a-hydrogen and facilitates proton
  abstraction. The product is an extended
  conjugated system incorporating both an imine and
  an enamine.

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• In the final step, protonation occurs at the
  d-carbon to the pyridine nitrogen, thus restoring
  the aromatic system. Hydrolysis of the imine
  gives the final products.

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Decarboxylation and PLP

• Decarboxylation reactions are important in
  biological systems because intermediates which
  are chemically disposed for decarboxylation, such
  as b-keto acids, occur frequently in primary and
  secondary metabolism.

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• a-Keto acids are chemically not predisposed
  towards decarboxylation. This is reflected in
  much higher temperatures required to effect the
  above transformation. Nature uses enzymes for
  this reaction which carry PLP as co-factor. The
  schemes below shows the decarboxylation of an
  a-amino acid.

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• The amino acid is bound to to PLP as the imine in
  the first step.

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• In the actual decarboxylation step, the
  electronic effects are the same the pyridine
  nitrogen acts as an electron-withdrawing group,
  this time facilitating deprotonation of the
  carboxylic acid group. Loss of carbondioxide and
  hydrolysis of the imine gives the reaction
  products.

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Transamination Reactions
a-Keto acid
a-Amino acid
a-Amino acid
a-Keto acid
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Transamination Reactions

• Most common amino acids can be converted into the
  corresponding keto acid by transamination
• This reaction swaps the amino group from one
  amino acid to a different keto acid, thereby
  generating a new pairing of amino acid and keto
  acid
• There is no overall loss or gain of nitrogen from
  the system

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Transamination Reactions, Contd

• Transamination reactions are readily reversible,
  and the equilibrium constant is close to 1
• One of the two substrate pairs is usually Glu and
  its corresponding keto acid a-KG

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Transamination Reactions, Contd

• The effect of transamination reactions is to
  collect the amino groups from many different
  amino acids in the form of L-Glu
• The Glu then functions as an amino group donor
  for biosynthetic pathways or for excretion
  pathways that lead to the elimination of
  nitrogenous waste products

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Transamination Reactions, Contd

• The substrates bind to the enzyme active center
  one at a time, and the function of the pyridoxal
  phosphate is to act as a temporary store of amino
  groups until the next substrate comes along
• In the process the pyridoxal phosphate is
  converted into pyridoxamine phosphate, and then
  back again Enzymologists call this a ping pong
  mechanism

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Transamination Reactions, Contd

• The condensation between the a-amino group and
  the aromatic aldehyde to form a Schiff base makes
  the a-carbon atom chemically reactive, so the
  isomerization of the Schiff base takes place very
  easily
• Many of the enyzmes that metabolize amino acids
  require PLP as a cofactor
• Unexpectedly, this compound also serves in a
  completely different manner in the active center
  of glycogen phosphorylase

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• Comparison of the active sites of L-aspartate
  aminotransferase (left) and D-amino acid
  aminotransferase (right)

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• The three-dimensional structures of bacterial
  D-amino acid aminotransferase (top) and human
  mitochondrial branched-chain L-amino acid
  aminotransferase (bottom)

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• Aspartate Transaminase (Aspartate
  Aminotransferase)

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Aspartate Transaminase

• Aspartate transaminase (AST) also called serum
  glutamic oxaloacetic transaminase (SGOT) or
  aspartate aminotransferase (ASAT/AAT) (EC
  2.6.1.1) is similar to alanine transaminase (ALT)
  in that it is another enzyme associated with
  liver parenchymal cells
• PLP coenzyme provides an aldehyde group to the
  enzyme, which is not available among the side
  chains of the 20 amino acids found in proteins

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Aspartate Transaminase, Contd

• The phosphate group provides a way to bind the
  coenzyme to the enzyme via a strong ionic
  interaction
• The aldehyde group readily reacts with primary
  amines like the a-amino groups of amino acids
• This process activates the amino group so that it
  can be cleaved by water

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Aspartate Transaminase, Contd

• This releases the keto-acid core of the amino
  acid and leaves the amino group on the enzyme
• Now the acceptor keto-acid binds and reacts with
  the activated amino group to form the new amino
  acid

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Aspartate Transaminase, Contd

• The mitochondrial aspartate transaminase provides
  an especially well studied example of PLP as a
  coenzyme for the transamination reactions
• The results of X-ray crystallographic studies
  provided detailed views of how PLP and substrates
  are bound and confirmed much of the proposed
  catalytic mechanism

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Aspartate Transaminase, Contd

• The enzyme is a dimer if identical subunits and
  it consists of a large domain and a small one
• PLP is bound to the large domain, in a pocket
  near the subunit interface
• In the absence of substrate, the aldehyde group
  of PLP is in a Schiff base linkage with Lys-258
• Arg-386 interacts with the a-carboxylate group of
  the substrate, helping to orient the substrate
  appropriately in the active site

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Structure of Aspartate Transaminase

• The active site of enzyme includes PLP attached
  to the enzyme by Schiff base linkage with Lys-258
• Arg-386 residue in the active site helps orient
  substrates by binding to their a-carboxylate
  groups

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Structure of Aspartate Transaminase

• Schematic diagram of the active site of E. coli
  aspartate aminotransferase
• Substrate specificity for the negatively charged
  aspartic acid substrate is determined by the
  positively charged guanidino groups of Arg-386
  and Arg-292, which have no catalytic role
• Mutation of Arg-292 to Asp produces an enzyme
  that prefers Arg to Asp as a substrate

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Stereochemistry of Aspartate Transaminase Reaction

• PLP enzymes cleave one of three bonds at the Ca
  atom of amino acids
• For example, bond a is cleaved by
  aminotransferase, bond b by dehydrogenase, and
  bond c by aldolase
• How can the same amino acid-PLP Schiff base be
  involved in the cleavage of the different bonds
  to an amino acid Ca?

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Stereochemistry of Aspartate Transaminase
Reaction, Contd

• For electrons to be withdrawn into the conjugated
  ring system of PLP, the p-orbital system of PLP
  must overlap with the bonding orbital containing
  the electron pair being delocalized
• This is possible only if the bond being broken
  lies in the plane perpendicular to the plane of
  the PLP p-orbital system
• Different bonds to Ca can be placed in this plane
  by rotation about the Ca-N bond

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Stereochemistry of Aspartate Transaminase
Reaction, Contd

• Each enzyme specifically cleaves its
  corresponding bond because the enzyme binds the
  amino acid-PLP Schiff base adduct with this bond
  in the plane perpendicular to that of the PLP
  ring
• This is an example of stereoelectronic assistance
  (effect)
• The enzyme binds substrate in a conformation that
  minimizes the electronic energy of the transition
  state

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Stereochemistry of Aspartate Transaminase
Reaction, Contd

• Bond orientation in a PLPamino acid Schiff base
• The ?-orbital framework of a PLPamino acid
  Schiff base
• The bond to Ca in the plane perpendicular to the
  PLP p-orbital system

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Stereochemistry of Aspartate Transaminase
Reaction, Contd

• In PLP-dependent transaminases active site, the
  addition of H from Lys residue to the bottom
  face of the quinoid intermediate determines the
  L-configuration of the amino acid product
• The conserved Arg residue interacts with the
  a-carboxylate group and helps establish the
  appropriate geometry of the quinonid intermediate

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Mechanism of L-Configuration of Amino Acids
Produced
b
a
a
b
For determination of stereochemistry of amino
acid formed
Stabilized carbanion resonance
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Stereochemistry of Aspartate Transaminase
Reaction, Contd

• The orientation about the NH-Ca bond determines
  the most favored reaction catalyzed by
  PLP-dependent enzymes
• The bond that is most nearly perpendicular to the
  p orbital of the PLP electron sink is most easily
  cleaved

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Stereochemistry of Aspartate Transaminase
Reaction, Contd

• In PLP-dependent transaminases, Ca-H bond is most
  nearly perpendicular to the p orbital system and
  is cleaved
• In SHMT, a small rotation about N-Ca bond places
  the Ca-Cb bond perpendicular to the p system,
  favoring its cleavage

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• Mechanism of Aspartate Transaminase

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Reaction of Aspartate Transaminase
Asp Transaminase
L-Asp
OAA
L-Glu
a-KG
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Reaction of Aspartate Transaminase
OAA
L-Glu
L-Asp
a-KG
E-PLP
PLP-Asp PLP-OAA
PLP-a-KG PLP-Glu
E-PMP
E-PLP
Ping Pong Mechanism
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Active Site of Asp Transaminase
General base
Both carboxylate groups of Asp are bound by
electrostatic interactions to the active site
Arg-292 and Arg-386
External aldimine (PLP-Asp Schiff base)
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Mechanism of Asp Transaminase
Tetrahedral intermediate
PLP
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Mechanism of Asp Transaminase, Contd
PLP-Enzyme Schiff base (Enzyme aldimine)
Asp
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Mechanism of Asp Transaminase, Contd
Tetrahedral intermediate
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Mechanism of Asp Transaminase, Contd
Abstract a-carbon
PLP-Asp Schiff base (Asp aldimine)
Quinonoid
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Mechanism of Asp Transaminase, Contd
Kitimine
Tetrahedral intermediate
OAA
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Mechanism of Asp Transaminase, Contd
PMP
a-KG
Tetrahedral intermediate
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Mechanism of Asp Transaminase, Contd
Protonation at a-carbon
Kitimine
Glu aldimine
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Mechanism of Asp Transaminase, Contd
Enzyme aldimine (PLP-Enzyme Schiff base)
Tetrahedral intermediate
Glu
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Experimental Evidences for the Role of Lys-258,
Arg-385 and Arg-292

• By using site-directed mutagenesis techniques by
  replacing Lys-258 for Ala gives a completely
  inactive mutant enzyme
• Replacing Lys-258 for Cys, the mutant enzyme is
  similarly inactive, however, if this enzyme is
  alkylated with 2-bromoethylalanine an active
  enzyme is obtained which contains a thioether
  analog of Lys at the active site

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Experimental Evidences for the Role of Lys-258,
Arg-385 and Arg-292, Contd

• This enzyme has 7 of the activity of wild-type
  enzyme with a slightly shifted pH rate profile of
  enzymatic activity
• Since the thioether-containing Lys analog is
  slightly less basic than Lys
• By replacing Arg-292 by other amino acids,
  mutation of Arg-292 to Asp-292 gave an enzyme
  whose catalytic efficiency for L-Asp has dropped
  from 34500 to 0.07 M-1s-1

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Experimental Evidences for the Role of Lys-258,
Arg-385 and Arg-292, Contd

• However, mutant enzyme was found to be capable of
  processing L-amino acid substrates containing
  positively charged side chains (Arg, Lys, and
  ornithine) which would interact favorably with
  Asp-292 with kcat/Km of 0.43 M-1s-1