Biosynthesis of penicillin in vitro: part II--purification and properties of 6-aminopenicillanic acid--phenylacetyl-CoA/phenoxyacetyl-CoA transferase
Kogekar, R.G.; Deshpande, V.N.
Indian Journal of Biochemistry and Biophysics 20(4): 208-212
1983
ISSN/ISBN: 0301-1208 PMID: 6423522 Document Number: 219865
Enzyme activities from the mycelia of Penicillium chrysogenum producing penicillin-G and penicillin-V were isolated, purified and characterized using the same procedure. 6-Aminopenicillanic acid and phenylacetyl-CoA/phenoxyacetyl-CoA are absolutely essential for the enzyme reaction. Mg2+ is a specific cation, dependence on this being 87% of normal reaction. Both enzyme activities have the same optimum pH (7.0), optimum temperature (32.degree. C) and Km (3.6 .times. 10-8 M). the CoA-derivatives of .beta.-substituted acetic acids, phenylacetyl-CoA and phenoxyacetyl-CoA, were equally efficient as substrates for each of the enzyme preparation. That p-methylphenoxyacetyl-CoA was only 20% as effctive as phenylacetyl-CoA/phenoxyacetyl-CoA indicated that any substitution in the phenyl ring reduced the enzyme activity. L-.alpha.-Aminodipyl-CoA was ineffective as substrate. Thiol-reagents, mercaptoethanol, dithiothreitol and reduced glutathione stimulated enzyme activity and N-ethylmaleimide and p-chloromercurobenzoate inhibited it. These observations along with the migration of the single protein band in gel tubes layered with individual enzyme preparation suggested that the 2 enzyme activities are the same.