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Substrate Stereochemistry of the Hydroxymethylglutaryl-CoA Lyase and Methylglutaconyl-CoA Hydratase Reactions

1975; Wiley; Volume: 53; Issue: 1 Linguagem: Inglês

10.1111/j.1432-1033.1975.tb04064.x

1432-1033

Burkhard Meßner, Hermann Eggerer, J. W. Cornforth, R. Mallaby,

Microbial Metabolic Engineering and Bioproduction

1 Two specimens of stereospecifically tritiated 3-hydroxy-3-methylgutaric acid, respectively (2S,3S) + (2R,3R) [2-3H1] and (2R,3S+ (2S,3R) [2-3H1], were prepared by oxidation of corresponding 4-tritiated mevalonates. 2 After being mixed with 3-hydroxy-3-methyl[3-14C]glutaric acid these specimens were converted chemically into two specimens of 3-hydroxy-3-methylglutaryl-coenzyme A, in which the enzymically active components were respectively (2S, 3S,4R)-[2,4-3H2,3-14C] (specimen I) and (2R,3S,4S)-[2,4-3H2,3-14C](specimen II). 3 Each specimen of 3-hydroxy-3-methylglutaryl-coenzyme A was subjected, in a deuterium oxide medium, to the action of hydroxymethylglutaryl-CoA lyase. Glyoxylate and malate synthase were also present and they converted the liberated deuterio-tritio-acetyl-coenzyme A into 2S-malate, which was isolated. 4 The two specimens of 2S-malate were examined for loss of tritium on incubation with fumarase. Malate from specimen I lost 73% and malate from specimen II lost 23%, of their tritium. 5 It was concluded that chiral acetyl-coenzyme A (S from I and R from II) was generated by the action of the lyase which was therefore stereospecific and proceeded with inversion of configuration. 6 Each specimen of 3-hydroxy-3-methylglutaryl-coenzyme A was subjected to the action of methylglutaconyl-CoA hydratase. The 3-methylglutaconyl-CoA produced by this action was converted chemically into 2, 3-dibromo-3-methylglutaconic acid which was examined for loss of tritium. 7 It was found that the glutaconic acid from specimen I retained 95–97% of the tritium present in the precursor, whereas the glutaconic acid from specimen II retained 47–50% of precursor tritium. 8 It was concluded that the action of methylglutaconyl-CoA hydratase is stereospecific and that addition-elimination of water is syn, not anti. 9 Further evidence is produced that the 3-methylglutaconyl-CoA substrate for this enzyme is geometrically the E-from. It was shown that when E-3-methylglutaconyl-CoA is hydrolysed by cold dilute alkali the E-3-methylglutaconic acid produced has suffered no exchange of hydrogen at the C-2 and C-4 positions but that the accompanying Z-3-methylglutaconic acid has suffered extensive exchange at both positions. An explanation is proposed.