Decarboxylations

1. 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

2. Decarboxylations Enzymes can catalyze the decarboxylation of both keto acids and hydroxy acids acetoacetate decarboxylase isocitrate dehydrogenase pyruvate decarboxylase malic enzyme

3. 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

4. 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

5. 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 ?

6. 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

7. Acetoacetate Decarboxylase catalytic mechanism Biochemistry35, 41 (1996)

8. 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

9. 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)

10. 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)

11. 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

12. 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 ?

13. 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

14. 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

15. 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

16. 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

17. Pyruvate Decarboxylase structure the enzyme is a tetramer composed of four identical subunits each monomer contains a bound TPP cofactor and a Mg2+ ion

18. Pyruvate Decarboxylase structure each subunit consists of three domains: pyruvate binding domain TPP domain regulatory domain J. Biol. Chem. 273, 20196 (1998)

19. 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)

20. 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

21. 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

22. 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

23. 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)

24. 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)

25. 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)

26. 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)

27. 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)

28. 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)