Purification and properties of isopenicillin N epimerase from Streptomyces clavuligerus.
Isopenicillin N epimerase, which catalyzes conversion of isopenicillin N to penicillin N, has been purified to electrophoretic homogeneity from the cell-free extract of Streptomyces clavuligerus by a procedure involving ammonium sulfate fractionation and chromatographies with DE-52, DEAE Affi-gel blue, Sephadex G-200, calcium phosphate-cellulose, and Mono Q. The purified epimerase is monomeric with a molecular weight of 47,000 or 50,000 as estimated by SDS-polyacrylamide gel electrophoresis or gel filtration, respectively. The enzyme contains 1 mol of pyridoxal 5'-phosphate per mol of protein, and shows absorption maxima at 280 and 420 nm. The epimerase catalyzes the complete 'racemization' on both the L-alpha-aminoadipyl side-chain of isopenicillin N and the D-alpha-aminoadipyl side-chain of penicillin N, so that an approximately equimolar mixture of the two penicillins is produced. The mixture is not truly racemic, since these penicillins are diastereomers rather than optical isomers. The chemical modification of primary amino groups of the epimerase by fluorescamine results in a great loss of the enzyme activity. The activity of purified enzyme is partially stimulated by the addition of sulfhydryl compounds. The activity is strongly inhibited by sulfhydryl group modifiers such as p-chloromercuribenzoate and N-ethylmaleimide.
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Department of Biochemistry, OAES, Oklahoma State University, Stillwater 74078.
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D000591:Amino Acid Isomerases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001426:Bacterial Proteins; D002384:Catalysis; D011232:Chemical Precipitation; D002845:Chromatography; D004789:Enzyme Activation / Q000187:drug effects; D005450:Fluorescamine / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010406:Penicillins / Q000378:metabolism; D011732:Pyridoxal Phosphate / Q000032:analysis; D013053:Spectrophotometry; D013302:Streptomyces / Q000201:enzymology*
D000596:Amino Acids; D001426:Bacterial Proteins; D010406:Penicillins; D005450:Fluorescamine; D011732:Pyridoxal Phosphate; D000591:Amino Acid Isomerases; C040886:isopenicillin N epimerase; C006414:penicillin N