Acetoacetate Decarboxylase

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Acetoacetate Decarboxylase

• In the presence of b-18Oacetoacetate, but in
  H216O, all of the 18O is released as H2O. This
  reaction is dependent on enzyme.
• When the reaction is run in the presence of
  unlabeled acetoacetate, but in H218O, the acetone
  generated contains 18O in the carbonyl.
• When the reaction is carried out in 2H2O,
  deuterium is incorporated into acetone. It is
  necessary to run this reaction with large amounts
  of enzyme for SHORT time periods. WHY?
• The pH optimum of the enzyme from Clostridium
  acetobutylicum is 5.9, and the pKa of the
  active-site lysine (K-115) was determined to be
  5.95, which is 4.5 pKa units lower than that of
  lysine in solution.

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Deuterated Water?
Enzyme isolated from Klebsiella aerogenes
catalyzes reaction with inversion of
stereochemistry to give (R)-acetoin from
(S)-a-acetolactate
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4-Oxalocrotonate Decarboxylase

• 4-Oxalocrotonate utilizes a metal cofactor (Mn2
  or Mg2) in the decarboxylation of a vinylogous
  b-keto acid.
• The first two intermediates were shown to be
  present in the reaction with the aid of
  specifically labeled substrate and UV and NMR
  spectroscopy.
• (5S)-5-2H1 is converted by 4-oxalocrotonate
  decarboxylase to (4E)-5-2H2.
• In the presence of D2O, incubation of 2 with the
  enzyme gives (3S)-3-2H3.
• The loss of CO2 and the incorporation of
  deuterium occur on the same side of the dienol
  intermediate.

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Cofactor lacking N-1 or N-4 nitrogens are
inactive, whereas the cofactor lacking the N-3
nitrogen is active.
From crystal structure of transketolase, and
biochemical studies of pyruvate decarboxylase, it
appears that a highly conserved glutamate is
important in deprotonation of cofactor. However,
it is not the actual base.
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Acetolactate Synthase
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Yeast Pyruvate Decarboxylase
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Pyruvate Dehydrogenase Complex
In aerobic organisms, pyruvate is not converted
into acetaldehyde and CO2. It is oxidatively
decarboxylated to give CO2 and acetyl CoA, which
is used in a number of metabolic reactions, and
which is an important substrate in the TCA cycle.
The pyruvate dehydrogenase complex is one of
several oxo-acid dehydrogenase complexes. Others
include the a-ketoglutarate dehydrogenase complex
(TCA cycle), the branched-chain oxo-acid
dehydrogenase complex, and the glycine cleavage
system. These complexes are huge (2-4 million
Da), and composed of multiple copies of several
different subunits. The E1 subunit contains
thiamin diphosphate and decarboxylase activity.
The E. coli protein contains 12 E1 dimers
arranged on the 12 edges of the E2 core. The E2
subunit is the core of the complexes, and
contains the cofactor, lipoic acid, which is
covalently bound via an amide linkage with a
conserved lysine residue, and which shuttles
intermediates from one subunit to the other. the
E3 subunit is a flavoprotein with
dihydrolipoamide dehydrogenase activity. The E.
coli complex contains 6 E3 dimers arranged on the
6 faces of the E2 core.
E. coli, 24 copies of E2 subunit in cubic symmetry
mammalian cells, 60 copies of E2 subunit in
a pentagonal dodecamer
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Cofactors in Oxo-acid Dehydrogenase Complexes
PDH Complex
Lipoic Acid (E2 subunit)
FAD (E3 subunit)
TDP (E1 subunit)
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Pyruvate Dehydrogenase Mechanism
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PDH-Acetyl Transferase
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PDH-Dihydrolipoamide Dehydrogenase
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Iron-Sulfur Clusters
Iron-sulfur cluster-containing proteins are
frequently used as electron transfer agents.
Since they can accept or donate only 1 electron,
they will typically reduce or oxidize proteins
that contain flavin cofactors, which can
stabilize one-electron intermediates.
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Pyruvate Ferredoxin Oxidoreductase
Pyruvate ferredoxin oxidoreductase is another
enzyme that can initiate the catabolism of
pyruvate and produced acetyl CoA. There are
clear similarities between this enzyme and the
PDH however, they differ substantially in the
nature of the intermediate electron acceptor.
These enzymes do not contain lipoyl cofactors,
but use iron-sulfur clusters to effect oxidative
decarboxylations. The enzyme is found in all
three kingdoms (Archaea, Bacteria, Eukaryotes).
It was apparently designed to work in organisms
that carry out anaerobic fermentation, since it
is not found in organisms that contain
mitochondria.
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Decarboxylation of Amino Acids

• Pyridoxal-5-phosphate is the coenzyme form of
  vitamin B6. Vitamin B6 refers to any of a group
  of related compounds lacking the phosphoryl
  group, including pyridoxal (CHO), pyridoxamine
  (-CH2NH2), and pyridoxine (-CH2OH).
• The cofactor is involved in a number of
  transformations involving amino acids, which
  include transaminations, a-decarboxylations,
  racemizations, a,b, eliminations, b,g
  eliminations, aldolizations, and the b
  decarboxylation of aspartic acid.
• In almost all cases, pyridoxal 5-phosphate
  promotes these reactions by stabilizing the
  resulting electron pairs at the a- or b-carbon
  atoms of a-amino acids.
• The cofactor is typically bound covalently as a
  Schiffs base with a conserved active-site lysine
  residue. The protonated form of the Schiffs
  base displays an absorbance in the visible region
  of the UV-vis spectrum.
• By forming a Schiffs base, the cofactor is
  activated for transimination, or formation of the
  external aldimine intermediate.

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Ornithine Decarboxylase
Ornithine decarboxylase catalyzes the
decarboxylation of the amino acid ornithine,
which is the key step in the synthesis of
polyamines. Polyamines are necessary components
of nearly all cells, from bacterial cells to
those of plants and humans. Regulation of the
enzyme occurs at the levels of transcription,
translation, and protein activity. The enzyme is
a dodecamer, composed of 6 dimers arranged in C6
symmetry.
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Structure of the Dimeric Unit of Ornithine
Decarboxylase
Pyridoxal phosphate shown in ball and stick model.
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Active Site of Ornithine Decarboxylase
Usually, in decarboxylases there are at least two
conserved amino acids. One is the lysine that
forms the internal aldimine. The other is an
aspartic or glutamic acid that regulates
protonation of the pyridine nitrogen
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Ornithine Decarboxylase Mechanism