On the mechanism and some properties of vinylacetyl-CoA delta-isomerase of Clostridium kluyveri.
Vinylacetyl-CoA delta-isomerase from Clostridium kluyveri grown on ethanol/acetate was purified 32-fold. The enzyme is rather labile. All experiments were conducted with the substrate analog thioester of vinylacetic acid and N-acetylchysteamine (vinylacetyl-SEtNAc (1 f)). 3-Butinoyl-SEtNAc is a strong inhibitor of the isomerase. The hydrogen transfer from the alpha-position of vinylacetyl-SEtNAc to the gamma-position of 2-butenoyl-SEtNAc (1f leads to 2 f) occurs partially intramolecularly (40-50%) as shown by experiments in 3HOH/H2O, 2H2O and 3HOH/2H2O as well as by experiments with [2,3-3H]vinylacetyl-SEtNAc. Only 0.07 atoms of tritium are incorporated into the gamma-position of 2f when the isomerisation takes place in 3HOH/H2O. The extent of intramolecularity is in agreement with results of experiments conducted in 2H2O with whole cells [4]. The reaction 1f leads to 2f shows no or only negligiebl reversibility and at least no considerable isotope effect.
On the mechanism and some properties of vinylacetyl-CoA delta-isomerase of Clostridium kluyveri. Vinylacetyl-CoA delta-isomerase from Clostridium kluyveri grown on ethanol/acetate was purified 32-fold. The enzyme is rather labile. All experiments were conducted with the substrate analog thioester of vinylacetic acid and N-acetylchysteamine (vinylacetyl-SEtNAc (1 f)). 3-Butinoyl-SEtNAc is a strong inhibitor of the isomerase. The hydrogen transfer from the alpha-position of vinylacetyl-SEtNAc to the gamma-position of 2-butenoyl-SEtNAc (1f leads to 2 f) occurs partially intramolecularly (40-50%) as shown by experiments in 3HOH/H2O, 2H2O and 3HOH/2H2O as well as by experiments with [2,3-3H]vinylacetyl-SEtNAc. Only 0.07 atoms of tritium are incorporated into the gamma-position of 2f when the isomerisation takes place in 3HOH/H2O. The extent of intramolecularity is in agreement with results of experiments conducted in 2H2O with whole cells [4]. The reaction 1f leads to 2f shows no or only negligiebl reversibility and at least no considerable isotope effect.