A spectrophotometric assay for deacetoxycephalosporin C synthase |
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Authors: | J E Baldwin M J Crabbe |
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Institution: | The Dyson Perrins Laboratory, University of Oxford, South Parks Road, Oxford OX1 3QY, England |
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Abstract: | A continuous direct spectrophotometric assay for deacetoxycephalosporin C synthase was developed, based on the absorption at 260 nm characteristic of the dihydrothiazine moiety of cephalosporins. Km values of 0.18 mM for penicillin N and 0.16 mM for alpha-ketoglutarate were determined. A coupled assay using succinate thiokinase, pyruvate kinase and lactate dehydrogenase showed that succinate was a product of both deacetoxycephalosporin C synthase and hydroxylase reactions. The expandase reaction exhibited a 1:1.06 stoichiometry for deacetoxycephalosporin C and succinate. |
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