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A spectrophotometric assay for deacetoxycephalosporin C synthase
Author(s) -
Baldwin Jack E.,
Crabbe M.James C.
Publication year - 1987
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(87)80087-x
Subject(s) - pyruvate kinase , chemistry , biochemistry , atp synthase , enzyme , chromatography , stereochemistry , glycolysis
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. K m values of 0.18 mM for penicillin N and 0.16 mM for α‐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.