1. During fermentation with whole cells of Acidaminococcus fermentans or Clostridium microsporum the pro-3S
hydrogen of (R)-2-hydroxyglutarate or of its precursor (S)-
glutamate is eliminated stereospecifically. Since (E)-
glutaconate but not its Z isomer is fermented by whole cells or cell-free extracts of A. fermentans, the overall
dehydration of (R)-2-hydroxyglutarate to (E)-
glutaconate can be described as syn. 2. The fermentation of (E)-
glutaconate required acetyl phophate,
CoA and NAD, that of (S)-
glutamate or (R)-2-hydroxyglutarate additionally
MgCl2, FeSO4 and
dithioerythritol. The fermentations of all three substrates were inhibited by
avidin and stimulated by
biotin. 3. The hydration of (E)-
glutaconate was measured enzymically by the formation of (R)-2-hydroxyglutarate. The
dehydration of the
hydroxy acid was assayed by the release of 3HOH from (2R)-2-hydroxy[3-3H]
glutarate. Optimum conditions were found by activation of the cell-free extract with
MgCl2, FeSO4,
dithioerythritol,
acetyl phosphate anmd
NADH followed by the reaction which only required
acetyl phosphate and CoA as cofactors. Activation and reaction had to be performed anaerobically. 4. The
dehydration was inhibited by 2 mM
azide, 1 mM
arsenate, 1 mM
hydroxylamine, 20 micro M dinitrophenol or 10 micro M
carbonylcyanide p-trifluoromethoxyphenylhydrazone. 5. It is concluded that the actual substrates of the
dehydration are the corresponding
thiol esters. The data indicate a catalytical phosphorylation during the reaction.