Condensations

1. Condensations Condensation reactions involve the formation of new carbon-carbon bonds The most common condensation reactions are between two aldehydes or ketones (aldol condensation) or between an ester and either a ketone or an ester (Claisen condensation) Enzymatic catalysis of these types of reactions involves the stabilization of the enolate form of the substrate for subsequent nucleophilic attack

2. Non-enzymatic Condensations aldol condensation Reaction of the α-carbon of an aldehyde or ketone with the carbonyl carbon of another aldehyde or ketone The nucleophile is formed by base catalyzed abstraction of the α-proton This carbanion attacks the electron deficient carbonyl carbon to form a new carbon-carbon bond

3. Non-enzymatic Condensations Claisen condensation Reaction of the α-carbon of an ester with the carbonyl carbon of another ester or ketone If there is a good leaving group in the ester then collapse of the tetrahedral carbon leads to an alcohol and a diketone

4. Aldol condensation acid/base catalysis The aldol condensation reaction is freely reversible in the presence of appropriate acid and base catalysts

5. Aldolases FDP aldolase deoxyribose-5-phosphate aldolase ketodeoxyphosphogluconatealdolase

6. Fructose diphosphatealdolase enzyme classes Aldolases fall into two general classes Class I: In animals and higher plants formation of an imine at the carbonyl group provides the “electron sink” for cleavage Class II: In prokaryotes this role is fulfilled by divalent metal ions human aldolase E. coli aldolase So here is a case where two enzymes catalyze exactly the same reaction with the same substrates, but use a different mechanism and have different structures

7. FDP Aldolase(class II) dimer structure zinc ion sodium ion Lys325 and Arg331

8. FDP Aldolase(class II) active site structure the catalytic zinc (Zn1) is trigonal bipyramidal the enzyme ligands to the metal are His110, His226 & His264 the metal also interacts with the hydroxyl and enolate oxygens of the T.S. analog PGH proposed transition state transition state analog

9. FDP Aldolase (class II) active site structure This analog (PGH) interacts with the Zn through the two oxygens the hydroxyl oxygen is also hydrogen bonded to Asp109 the phosphate group is bound to Ser267, Thr289 and Lys325

10. FDP Aldolase (class II) active site structure a model of substrate binding places DHAP in the same orientation as the PGH inhibitor glyceraldehyde-3-phosphate is proposed to bind adjacent to the DHAP binding site Arg331 is in position to interact with the phosphate group of G3P Asp288 and Asp109 can bind to the oxygens of G3P

11. FDP Aldolase (class II) catalytic mechanism Removal of a proton by Glu182 generates the enolate nucleophile Attack on the aldehyde forms the new carbon-carbon bond Proton transfer from Asp109 completes the reaction J. Molec. Biol.287, 383 (1999)

12. FDP Aldolase (class I) proposed mechanism protonated Schiff base can act as an electron sink dehydration and Schiff base formation attack by active site lysine nucleophile proton abstraction catalyzes bond cleavage hydrolysis releases the other product resonance stabilization of the carbanion intermediate G3P DHAP

13. Deoxyribose5P Aldolase (class I) structure apo-WT carbinolamine complex the enzyme has a b-barrel structure J. Mol. Biol.343, 1019 (2004)

14. KDPG Aldolase Catalyzes the reversible aldol condensation of pyruvate and glyceraldehyde-3-phosphate to produce 2-keto-3-deoxy-6-phosphogluconate (KDPG) For a class I aldolase a covalent Schiff base intermediate is formed during the catalytic cycle These enzymes form a Schiff base without using pyridoxal phosphate or quinones as cofactors

15. KDPG Aldolase proposed mechanism Proc. Natl. Acad. Sci.98, 3679 (2001)

16. KDPG Aldolase Schiff base intermediate there is continuous electron density between the lysine and pyruvate that confirms the identity of the Schiff base Proc. Natl. Acad. Sci.98, 3679 (2001)

17. KDPG Aldolase Schiff base intermediate the adduct is stabilized by interactions with each of the oxygens of pyruvate Proc. Natl. Acad. Sci.98, 3679 (2001)

18. Class I Aldolases active sites • DER aldolase • FBP aldolase C. FBP aldolase complex D. KDPG aldolase complex E. transaldolase complex F. ALA dehydratase complex Each enzyme has a β-barrel structure with a lysine positioned in the active site for Schiff base formation J. Mol. Biol.343, 1019 (2004)

19. Enzyme-catalyzed Claisen Condensations citrate synthase citrate lyase What is the major difference between these two reactions ?

20. Claisen condensation transport of acetyl groups These two enzymes, citrate synthase and citrate lyase, function together to catalyze the same reaction but in opposite directions The net effect is to transport an acetyl group across the mitochondrial membrane The mitochondrial acetyl-CoA formed from these reactions is the precursor for fatty acid biosynthesis

21. Citrate synthase reaction Because a thioester is a higher energy compound the production of citrate is an energetically favorable reaction the proposed reaction intermediate is citryl-CoA Biochemistry29, 2213 (1990)

22. Citrate synthase structure Coenzyme A andoxaloacetateare bound in the active site of citrate synthase Biochemistry29, 2213 (1990)

23. Citrate synthase active site structure There are numerous interactions between the enzyme and coenzyme A All of the functional groups of citrate are also involved in hydrogen bonding interactions

24. Citrate synthase catalytic mechanism the enol attacks the carbonyl of OAA His274 assists by deprotonation Asp375 is the catalytic base that abstracts a proton His274 acts as an acid to protonate the carbonyl oxygen the resulting citryl-CoA intermediate must be hydrolyzed to yield the final product

25. Citrate lyase reaction citrate lyase catalyzes the reverse reaction of citrate synthase, the transfer of an acetyl group from citrate to coenzyme A because the reaction in this direction is energetically unfavorable a different enzyme is used with a different catalytic mechanism citrate lyase uses the energy of ATP to drive this Claisen cleavage reaction

26. Citrate lyase catalytic mechanism the initial step involves the phosphorylation of a group on the enzyme attack by citrate results in phosphoryl transfer to activate the substrate attack by the active site nucleophile displaces phosphate and produces a covalent citryl-enzyme intermediate

27. Citrate lyase catalytic mechanism binding of CoA followed by thiolate attack displaces the citrate to form citryl-CoA proton abstraction catalyzes the Claisen cleavage reaction yielding the acetyl-CoA and oxaloacetate products

28. Condensations • condensation reactions are important bond forming reactions • aldolases catalyze the reversible condensation between aldehydes or ketones using two different mechanisms • Class I aldolases use an active site lysine to form a Schiff base intermediate in plants and animals • Class II aldolases in microorganisms catalyze this reaction using a divalent metal ion • Claisen condensations can be coupled for the transport of groups across membranes