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 language | English |
|---|---|
| Pages (from-to) | 3719-3723 |
| Number of pages | 5 |
| Journal | Biochemistry |
| Volume | 20 |
| Issue number | 13 |
| Publication status | Published - 1981 |
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- 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 journal › Article
}
TY - JOUR
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
PY - 1981
Y1 - 1981
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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M3 - Article
C2 - 7023536
AN - SCOPUS:0019886922
VL - 20
SP - 3719
EP - 3723
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 13
ER -