The discovery that the L-serine dehydratase (E.C. 4.2.1.13) from Escherichia coli B could be stabilised by iron and dithiothreitol has allowed this enzyme to be purified to a level approaching homogeneity. The purification scheme involved chemical treatments with streptomycin sulphate and ammonium sulphate, and elution from DEAE cellulose, pentyl agarose. Mono Q, Reactive Green and Phenyl superose chromatography columns. The purified dehydratase had a specific activity of 1653 ?moles of pyruvate min-l mg-1 enzyme and was, as judged by SDS PAGE, about 90 % pure. Overall recoveries were in the region of 4 % of the starting activity. A series of spectroscopic investigations and inhibitor studies showed that L- serine dehydratase did not utilise pyridoxal phosphate as cofactor. However, the purified enzyme did show an absolute requirement for iron and dithiothreitol for activity. The activation produced by these reagents was characterised and found to be slow, markedly influenced by both temperature and pH, and could be prevented, or reversed, by metal chelators, such as EDTA and o-phenanthroline. The activation process was also oxygen-dependent, and appeared to involve the production of an oxygen radical, since it was subject to inhibition by catalase and stimulation by hydrogen peroxide. Activation of L-serine dehydratase by iron and DTT also appeared to involve iron binding, at a ratio of 2 - 3 ?moles of Fe per ?mole enzyme. However, UV/visible and EPR investigations were unable to identify the structural form in which this bound iron existed. L-Serine dehydratase was found to be specific for L-serine; D-serine, L-threonine and L-cysteine were not deaminated. The timecourse of pyruvate formation was found to be non-linear, and the substrate saturation curve for L-serine sigmoidal, with an S[0.5] value of 2.6 mM, and a Hill coefficient of 2.13. The dehydratase could be activated by its substrate, L-serine, or substrate analogue, D-serine, which resulted in the production of a linear timecourse and hyperbolic substrate saturation profile (S[0.5] 2.8 mM, Hill coefficient 1.13). The molecular basis of this substrate activation process was investigated, and appeared to have its origins in a slow, serine-dependent rearrangement of the tertiary structure of the enzyme rather, than had previously been suggested from studies of the dehdyratase in crude extracts, a dimerisation reaction. In common with other microbial L-serine dehydratases, the purified E. coli B enzyme showed a broad pH optimum for pyruvate production, with maximal activity occurring between pH 7.8 and 8.2. It was inhibited competitively by L-cysteine and D-serine, with Ki values of 1.6 and 4.2 mM, respectively, and irreversibly by sulphydryl-active agents such as DTNB, N-ethylmaleimide and HgC12. In addition, the N-terminal amino acid sequence of the E. coli B L-serine dehydratase was analysed, and was found to show a high level of similarity with the predicted N-terminal sequence of the L-serine dehydratase from E. coli K12.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:674248 |
| Date | January 1992 |
| Creators | Freestone, Primrose P. E. |
| Publisher | University of Leicester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/2381/35225 |
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