The Stereochemistry at Carbon 3 of Pyruvate Lyase Condensation Products

Both the Schiff base-mediated 2-keto-4-hydroxyglutarate aldolase of liver and the metal-activated α-keto-β-deoxyhexarate aldolase of Escherichia coli condense pyruvate and glyoxylate to form (4-RS)-2-keto-4-hydroxyglutarate. Both enzymes catalyzed a condensation between [3-3H3]pyruvate and glyoxylate. It was found that C—C synthesis occurred 6 to 7 times faster than C—T bond cleavage. Thus, pyruvate deprotonation appeared to be partially rate-limiting in C—C synthesis. Condensations were carried out between (3R)-[3-3H, 2H, H]pyruvate or (3S)-[3-3H, 2H, H]pyruvate to form (4-RS)-2-keto-4-hydroxy-[3-3H, 2H]glutarate which was decarboxylated to dl-[3-3H, 2H]malate. The prochirality of malate's C-3 was analyzed by determining tritium release catalyzed by fumarase and maleate hydratase. It was found that malate derived from (3R)-[3-3H, 2H, H]pyruvate was of (3R)-3-3H, 2H-configuration while that derived from (3S)-[3-3H, 2H, H]pyruvate was of (3S)-3-3H, 2H-configuration. Comparison with stereochemical models shows that the configuration at C-3 of pyruvate is thus retained during C—C synthesis catalyzed by the two aldolases studied, i.e. both the exchanging proton and aldehyde approach the same face of bound enolpyruvate. A stereochemical mechanism explaining the synthesis of racemic condensation products is discussed.