Substrates and mechanism of 2-hydroxyglutaryl-CoA-dehydratase from Clostridium symbiosum
Loading...
Files
Date
Publisher
Philipps-Universität Marburg
Abstract
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.
Review
Metadata
License
This item has been published with the following license: In Copyright