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Decarboxylations. decarboxylases are one class of a diverse group of enzymes that can catalyze the synthesis and cleavage of carbon-carbon bonds The primary requirement to facilitate decarboxylation is the ability of the enzyme to stabilize the developing carbanionic transition state

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slide1

Decarboxylations

decarboxylases are one class of a diverse group of enzymes that can catalyze the synthesis and cleavage of carbon-carbon bonds

The primary requirement to facilitate decarboxylation is the ability of the enzyme to stabilize the developing carbanionic transition state

In simpler terms, we need a place to keep the electrons (an “electron sink”) during the cleavage of the carbon-carbon bond

slide2

Decarboxylations

Enzymes can catalyze the decarboxylation of both keto acids and hydroxy acids

acetoacetate decarboxylase

isocitrate dehydrogenase

pyruvate decarboxylase

malic enzyme

slide3

Decarboxylation of a β-keto acid

β-keto acids will frequently undergo spontaneous decarboxylation, with the reaction accelerated under acidic conditions

acid-catalyzed conversion of the enolate to a stable product

slide4

Decarboxylation of a β-keto acid

amine catalysis

formation of an intermediate imine

mechanism of catalysis

protonation provides an electron sink for decarboxylation

acid catalyzed rearrangement

hydrolysis will lead to the ketone product

slide5

Acetoacetate Decarboxylase

reaction and labeling pattern

If the carbonyl oxygen of acetoacetate is labeled (red dot)no label is found in the acetone product

If the reaction is run in 18O-water the label is then found in acetone (green dot)

If the reaction is run in 3H-water that label is also found in the product (green dot)

What does this labeling pattern tell us about the mechanism of the reaction ?

slide6

Acetoacetate Decarboxylase

proposed reaction intermediate

Hydrolysis of the Schiff base would lead to the introduction of the label

Reduction leads to trapping of the enzyme bound intermediate and identifies an active site lysine as the site of Schiff base formation

However, a pH profile study shows that the active site nucleophile of this enzyme has a pK value of about 6, which is much too low for the amine of a lysine

slide7

Acetoacetate Decarboxylase

catalytic mechanism

Biochemistry35, 41 (1996)

slide8

AcetoacetateDecarboxylase

active site mutants

Elimination of the catalytic Lys115 results in complete loss of activity

Chemical rescue with Cys-ethylamine recovers some activity

Elimination of Lys116 also leads to substantial activity loss

Replacement of Lys116 with another positively charged group (either Arg or Cys-EA) leads to some activity recovery

These results are consistent with the hypothesis that the presence of a positive charge at position 116 lowers the pK of the Lys115 nucleophile

slide9

AcetoacetateDecarboxylase

the latest update

The recently determined structure of this enzyme does not support this hypothesis !

Lys115 and Lys116 are pointing in opposite directions !

The charge on Lys116 is unlikely to affect the pK value of Lys115

So what is the explanation for the unusually low pK of Lys115 ?

there are only two charged amino acids in the active site and they are too far away to stabilize the positive charge

the hydrophobic environment of the active site destabilizes the protonated amine

The inhibitor 2,4-pentanedione is interacting with Arg29 & Glu76

This would orient the β-carbonyl of the substrate towards Lys115 and in position for Schiff base formation

Nature459, 393 (2009)

slide10

Isocitrate Dehydrogenase

overall structure

The enzyme is a dimer containing a bound divalent metal ion in each monomer

In this structure Ca2+ is bound to produce an inactive enzyme

The metal ion is immediately adjacent to the substrate binding site

J. Molec. Biol.295, 377 (2000)

slide11

Isocitrate Dehydrogenase

metal ion coordination

The Ca ion (and presumably the Mn ion) is coordinated by three carboxyl groups provided by aspartic acids

The metal is also within coordination distance to the substrate hydroxyl group

Two solvent water molecules complete the coordination shell

This metal ion binding site is in position to act as the electron sink that is required to catalyze decarboxylation

slide12

Isocitrate Dehydrogenase

active site structure

Isocitrate is bound by interactions with three arginines (R101, R110 & R133)

The carboxyl group that is removed is hydrogen-bonded to Y140 & K212

Three aspartates (D252, D275 & D279) form the divalent metal ion site

How does the enzyme catalyze the decarboxylation of the intermediate ?

slide13

Isocitrate Dehydrogenase

reaction and mechanism

Oxidation of the alcohol to a ketone leads to a keto acid for decarboxylation

But which carboxyl group will be eliminated ?

Decarboxylation of aβ-keto acid is much easier

oxidation to a keto acid

slide14

Isocitrate Dehydrogenase

decarboxylation reaction

After hydride transfer to form the keto acid the enzyme uses the divalent metal ion to promote the decarboxylation

The Mn ion stabilizes the developing negative charge on the carbonyl oxygen (electron sink)

isomerization and protonation of the bound enolate leads to the final product

slide15

A Decarboxylation Cofactor

Thiamine Pyrophosphate

Where is the critical functional group in this structure ?

This resonance stabilized carbanion can act as a nucleophile

Adduct formation with an α-keto acid will provide an electron sink for decarboxylation

slide16

Pyruvate Decarboxylase

catalytic reaction

This enzyme used a thiamin pyrophosphate cofactor to catalyze the decarboxylation of an α-keto acid to produce an aldehyde

Acetaldehyde can then be converted to ethanol by alcohol dehydrogenase

slide17

Pyruvate Decarboxylase

structure

the enzyme is a tetramer composed of four identical subunits

each monomer contains a bound TPP cofactor and a Mg2+ ion

slide18

Pyruvate Decarboxylase

structure

each subunit consists of three domains:

pyruvate binding domain

TPP domain

regulatory domain

J. Biol. Chem. 273, 20196 (1998)

slide19

Pyruvate Decarboxylase

TPP cofactor binding

Mg2+ ion coordinates to the pyrophosphate

hydrogen bonds position the pyrimidine ring

the active thiazole ring is held only by hydrophobic interactions

two water molecules occupy the pyruvate binding site

the catalytic His113 undergoes a conformational shift

J. Biol. Chem. 273, 20196 (1998)

slide20

Pyruvate Decarboxylase

catalytic intermediate

Glu473 is proposed to act as an acid/base catalyst

The interaction between Asp27 and His113 keeps histidine protonated

This keeps His114 uncharged to not interfere with the developing positive charge on the pyrimidine amino group during proton abstraction

slide21

Pyruvate Decarboxylase

catalytic mechanism

Deprotonation of TPP produces the active form of the cofactor

Attack on the carbonyl of pyruvate produces a covalent intermediate

Release of carbon dioxide leaves a resonance stabilized carbanion

Protonation of the intermediate allows its breakdown and release of the acetaldehyde product

slide22

Malic Enzyme

Catalyzes the oxidative decarboxylation of malate to pyruvate and CO2

This is an important reaction in the fixation of CO2 in plants

Malate carries CO2 across the cell membrane

Malic enzyme catalyzes the decarboxylation to release CO2

pyruvate then returns to pick up another CO2

slide23

Malic Enzyme

overall structure

The enzyme is a tetramer that is composed of a dimer of dimers

there are extensive subunit contacts in the horizontal direction

Only a few loops make contacts in the vertical direction

Protein Sci.11, 332 (2002)

slide24

Malic Enzyme

domain organization

Each subunit is organized into four domains

The enzyme is present in an open conformation when NAD is bound

When the substrate or an inhibitor binds the conformation closes to produce the catalytically active enzyme form

Enzyme complex with NADP, Mn, and the inhibitor oxalate

Protein Sci.11, 332 (2002)

slide25

Malic Enzyme

active site

Binding of NADP, Mn2+andmalate

Glu255, Glu256 and Asp279 provide metal binding ligands

The metal is in position to interact with both the carboxyl and carbonyl groups of the substrate

Interactions with the nicotinamide, ribose and phosphates help position the cofactor

Biochemistry42, 12728 (2003)

slide26

Malic Enzyme

proposed mechanism

substrate binding to M2+ and active site groups

hydride transfer to produce oxaloacetate intermediate

decarboxylation assisted by the divalent metal ion

rearrangement to obtain the pyruvate product

three separate transformations are each enzyme-catalyzed

Biochemistry42, 12728 (2003)

slide27

Malic Enzyme

coenzyme specificity

The liver enzyme is specific for NADP

while the mitochondrial enzyme uses NAD

Overlay of the NAD and NADP utilizing enzymes

Lys347 & Lys362 interact with the 2’-phosphate

These lysines are conserved in the NADP requiring enzymes but not in the NAD variety

Protein Sci.11, 332 (2002)

slide28

Decarboxylations

  • β-keto acids are readily decarboxylated (acetoacetate decarboxylase)
  • isocitrate dehydrogenase uses a divalent metal ion to promote decarboxylation after oxidation
  • α-keto acids require adduct formation with TPP (pyruvate decarboxylase)
  • β-hydroxy acids are oxidized to β-keto acids prior to decarboxylation (malic enzyme)