Stereochemical outcome of processing of fluorinated substrates by ATP citrate lyase and malate synthase

Michael A. Marletta, Paul A. Srere, Christopher Walsh

Research output: Contribution to journalArticle

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Abstract

The (-)-erythro- and (+)-erythro-2-fluorocitrate isomers are substrates for the cytoplasmic ATP citrate lyase from rat liver with Vmax values 0.13% and 2.3%, respectively, that of citrate when the keto acid products are measured by in situ reduction by NADH and malate dehydrogenase. Anticipated regiospecificity of cleavage by ATP citrate lyase is production of 2-fluoroacetyl-CoA and oxalacetate from a (2R,3R)-2-fluorocitrate isomer and production of acetyl-CoA and (3S)-fluorooxalacetate from cleavage of the other erythro diastereomer, the (2S,3S)-2-fluorocitrate. The product combinations were validated by in situ reduction with MDH and (4R)-[3H]NADH to yield L-(2S)-[2-3H] malate from (-)-erythro-2-fluorocitrate and to yield L-(2R,3S)-3-fluoro[2-3H] malate only from the (+)-erythro-2-fluorocitrate. The data allow for the first time unambiguous assignment of absolute stereochemistry to the (+)-erythro-2-fluorocitrate (2S,3S) and thereby necessarily to the (-)-erythro-2-fluorocitrate as 2R,3R. This latter isomer is, of course, the toxic species generated by the "lethal synthesis" from fluoroacetyl-CoA and oxalacetate effected by citrate synthase. Assuming inversion in the citrate synthase reaction, one can now state that it processes 2-fluoroacetyl-CoA chirally with specific pro-S hydrogen abstraction in the condensation. Cleavage of (2R,3R)-2-fluorocitrate by ATP citrate lyase in 3H2O should yield a product that is chiral, (2S)-fluoro[2-3H]acetyl-CoA. Attempts to analyze this product involved in situ coupling with [14C]-glyoxalate and yeast malate synthase. Surprisingly, both erythro- and threo-3-fluoromalates formed in about equal amounts, and each had both 3H and 14C radioactivity. These results suggest that malate synthase, in contrast to citrate synthase, processes one of its two substrates achirally at a prochiral center, either C2 of fluoroacetyl-CoA or, less likely, the trigonal aldehyde carbon of glyoxalate during fluoromalate biosynthesis.

Original languageEnglish
Pages (from-to)3719-3723
Number of pages5
JournalBiochemistry
Volume20
Issue number13
Publication statusPublished - 1981

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Malate Synthase
ATP Citrate (pro-S)-Lyase
Substrates
Processing
Citrate (si)-Synthase
Isomers
Acetyl Coenzyme A
Keto Acids
NADH Dehydrogenase
Malate Dehydrogenase
Stereochemistry
fluorocitrate
Poisons
Biosynthesis
Radioactivity
Aldehydes
Citric Acid
Liver
NAD
Yeast

ASJC Scopus subject areas

  • Biochemistry

Cite this

Stereochemical outcome of processing of fluorinated substrates by ATP citrate lyase and malate synthase. / Marletta, Michael A.; Srere, Paul A.; Walsh, Christopher.

In: Biochemistry, Vol. 20, No. 13, 1981, p. 3719-3723.

Research output: Contribution to journalArticle

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abstract = "The (-)-erythro- and (+)-erythro-2-fluorocitrate isomers are substrates for the cytoplasmic ATP citrate lyase from rat liver with Vmax values 0.13{\%} and 2.3{\%}, respectively, that of citrate when the keto acid products are measured by in situ reduction by NADH and malate dehydrogenase. Anticipated regiospecificity of cleavage by ATP citrate lyase is production of 2-fluoroacetyl-CoA and oxalacetate from a (2R,3R)-2-fluorocitrate isomer and production of acetyl-CoA and (3S)-fluorooxalacetate from cleavage of the other erythro diastereomer, the (2S,3S)-2-fluorocitrate. The product combinations were validated by in situ reduction with MDH and (4R)-[3H]NADH to yield L-(2S)-[2-3H] malate from (-)-erythro-2-fluorocitrate and to yield L-(2R,3S)-3-fluoro[2-3H] malate only from the (+)-erythro-2-fluorocitrate. The data allow for the first time unambiguous assignment of absolute stereochemistry to the (+)-erythro-2-fluorocitrate (2S,3S) and thereby necessarily to the (-)-erythro-2-fluorocitrate as 2R,3R. This latter isomer is, of course, the toxic species generated by the {"}lethal synthesis{"} from fluoroacetyl-CoA and oxalacetate effected by citrate synthase. Assuming inversion in the citrate synthase reaction, one can now state that it processes 2-fluoroacetyl-CoA chirally with specific pro-S hydrogen abstraction in the condensation. Cleavage of (2R,3R)-2-fluorocitrate by ATP citrate lyase in 3H2O should yield a product that is chiral, (2S)-fluoro[2-3H]acetyl-CoA. Attempts to analyze this product involved in situ coupling with [14C]-glyoxalate and yeast malate synthase. Surprisingly, both erythro- and threo-3-fluoromalates formed in about equal amounts, and each had both 3H and 14C radioactivity. These results suggest that malate synthase, in contrast to citrate synthase, processes one of its two substrates achirally at a prochiral center, either C2 of fluoroacetyl-CoA or, less likely, the trigonal aldehyde carbon of glyoxalate during fluoromalate biosynthesis.",
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T1 - Stereochemical outcome of processing of fluorinated substrates by ATP citrate lyase and malate synthase

AU - Marletta, Michael A.

AU - Srere, Paul A.

AU - Walsh, Christopher

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N2 - The (-)-erythro- and (+)-erythro-2-fluorocitrate isomers are substrates for the cytoplasmic ATP citrate lyase from rat liver with Vmax values 0.13% and 2.3%, respectively, that of citrate when the keto acid products are measured by in situ reduction by NADH and malate dehydrogenase. Anticipated regiospecificity of cleavage by ATP citrate lyase is production of 2-fluoroacetyl-CoA and oxalacetate from a (2R,3R)-2-fluorocitrate isomer and production of acetyl-CoA and (3S)-fluorooxalacetate from cleavage of the other erythro diastereomer, the (2S,3S)-2-fluorocitrate. The product combinations were validated by in situ reduction with MDH and (4R)-[3H]NADH to yield L-(2S)-[2-3H] malate from (-)-erythro-2-fluorocitrate and to yield L-(2R,3S)-3-fluoro[2-3H] malate only from the (+)-erythro-2-fluorocitrate. The data allow for the first time unambiguous assignment of absolute stereochemistry to the (+)-erythro-2-fluorocitrate (2S,3S) and thereby necessarily to the (-)-erythro-2-fluorocitrate as 2R,3R. This latter isomer is, of course, the toxic species generated by the "lethal synthesis" from fluoroacetyl-CoA and oxalacetate effected by citrate synthase. Assuming inversion in the citrate synthase reaction, one can now state that it processes 2-fluoroacetyl-CoA chirally with specific pro-S hydrogen abstraction in the condensation. Cleavage of (2R,3R)-2-fluorocitrate by ATP citrate lyase in 3H2O should yield a product that is chiral, (2S)-fluoro[2-3H]acetyl-CoA. Attempts to analyze this product involved in situ coupling with [14C]-glyoxalate and yeast malate synthase. Surprisingly, both erythro- and threo-3-fluoromalates formed in about equal amounts, and each had both 3H and 14C radioactivity. These results suggest that malate synthase, in contrast to citrate synthase, processes one of its two substrates achirally at a prochiral center, either C2 of fluoroacetyl-CoA or, less likely, the trigonal aldehyde carbon of glyoxalate during fluoromalate biosynthesis.

AB - The (-)-erythro- and (+)-erythro-2-fluorocitrate isomers are substrates for the cytoplasmic ATP citrate lyase from rat liver with Vmax values 0.13% and 2.3%, respectively, that of citrate when the keto acid products are measured by in situ reduction by NADH and malate dehydrogenase. Anticipated regiospecificity of cleavage by ATP citrate lyase is production of 2-fluoroacetyl-CoA and oxalacetate from a (2R,3R)-2-fluorocitrate isomer and production of acetyl-CoA and (3S)-fluorooxalacetate from cleavage of the other erythro diastereomer, the (2S,3S)-2-fluorocitrate. The product combinations were validated by in situ reduction with MDH and (4R)-[3H]NADH to yield L-(2S)-[2-3H] malate from (-)-erythro-2-fluorocitrate and to yield L-(2R,3S)-3-fluoro[2-3H] malate only from the (+)-erythro-2-fluorocitrate. The data allow for the first time unambiguous assignment of absolute stereochemistry to the (+)-erythro-2-fluorocitrate (2S,3S) and thereby necessarily to the (-)-erythro-2-fluorocitrate as 2R,3R. This latter isomer is, of course, the toxic species generated by the "lethal synthesis" from fluoroacetyl-CoA and oxalacetate effected by citrate synthase. Assuming inversion in the citrate synthase reaction, one can now state that it processes 2-fluoroacetyl-CoA chirally with specific pro-S hydrogen abstraction in the condensation. Cleavage of (2R,3R)-2-fluorocitrate by ATP citrate lyase in 3H2O should yield a product that is chiral, (2S)-fluoro[2-3H]acetyl-CoA. Attempts to analyze this product involved in situ coupling with [14C]-glyoxalate and yeast malate synthase. Surprisingly, both erythro- and threo-3-fluoromalates formed in about equal amounts, and each had both 3H and 14C radioactivity. These results suggest that malate synthase, in contrast to citrate synthase, processes one of its two substrates achirally at a prochiral center, either C2 of fluoroacetyl-CoA or, less likely, the trigonal aldehyde carbon of glyoxalate during fluoromalate biosynthesis.

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