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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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| Contributors: | |
| Format: | Doctoral Thesis |
| Language: | English |
| Published: |
Philipps-Universität Marburg
2009
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| Subjects: | |
| Online Access: | PDF Full Text |
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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. |
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| Physical Description: | 135 Pages |
| DOI: | 10.17192/z2009.0155 |