Substrates and mechanism of 2-hydroxyglutaryl-CoA-dehydratase from Clostridium symbiosum
Muconyl-CoA, 2-hydroxyadipoyl-CoA, oxalocrotonyl-CoA and butynedioyl- CoA were synthesised and characterised as substrates of the (R)-2- hydroxyglutaryl-CoA dehydratase from Clostridium symbiosum. The specificity of the enzyme for these substrates were determined and the reaction products identi...
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|Summary:||Muconyl-CoA, 2-hydroxyadipoyl-CoA, oxalocrotonyl-CoA and butynedioyl- CoA were synthesised and characterised as substrates of the (R)-2- hydroxyglutaryl-CoA dehydratase from Clostridium symbiosum. The specificity of the enzyme for these substrates were determined and the reaction products identified by MALDI-TOF mass spectrometry. 2,2-Difluoroglutaryl-CoA was synthesised and characterised as an inhibitor of the dehydratase activity (Ki = 0.069 mM). It could be shown that the inhibition of the dehydratase by metronidazole observed by earlier investigators was most likely due to the destruction of the iron-sulfur cluster of the activator (which is an accessory enzyme required to start the dehydratase catalysis). Further, lactyl-CoA dehydratase from Clostridium propionicum was assayed spectrophotometrically and purified to apparent homogeneity. A combination of kinetic experiments performed with (R)-2-hydroxyglutaryl-CoA dehydratase and lactyl-CoA dehydratase and their respective substrates, aand theoretical calculations showed that the chemical structure of the 2-hydroxyacyl-CoA had a large effect on the equilibrium constant of its conversion to 2-enoyl-CoA. Finally, two new substrates (oxalocrotonate and 2-hydroxyadipate), and a competitive inhibitor (2,2-difluoroglutarate, Ki = 0.62 mM) of the (R)-2- hydroxyglutarate dehydrogenase from Acidaminococcus fermentans were characterised. Modelling of these compounds into the active site of this enzyme supported the biochemical observations.|