Direct evaluation of phenylacetyl-CoA: 6-aminopenicillanic acid acyltransferase of Penicillium chrysogenum by bioassay.
The enzyme phenylacetyl-CoA: 6-Aminopenicillanic acid acyltransferase of Penicillium chrysogenum was evaluated by direct bioassay against Micrococcus luteus ATCC 9341. The enzyme required dithiothreitol, was inactivated by 0.2 mM Hg2+ (100%), Zn2+ (80%), Cu2+ (60%), 1 mM N-ethylmaleimide (80%), and showed maximal catalytic activity at pH 8.4 and 20 degrees C. The V50 values for phenylacetyl-CoA and 6-aminopenicillanic acid were 0.55 mM and 1 microM, respectively. When octanoyl-CoA was employed as substrate similar results were obtained. In both cases the product generated showed strong antibacterial activity which was quickly lost when incubation was carried out with beta-lactamase. Reactions performed in the presence of Escherichia coli penicillin acylase did not generated active products when phenylacetyl-CoA was the substrate; they did with octanoyl-CoA. Time-course experiments revealed that the highest enzyme levels are found in 36 hours mycelium and remained almost constant from 48 to 96 hours; thereafter the level of the enzyme slowly decreased.
D000105:Acetyl Coenzyme A / Q000031:analogs & derivatives / Q000378:metabolism; D000217:Acyltransferases / Q000378:metabolism*; D001681:Biological Assay; D004229:Dithiothreitol / Q000494:pharmacology; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D008837:Micrococcus / Q000187:drug effects; D010397:Penicillanic Acid / Q000378:metabolism / Q000031:analogs & derivatives; D010400:Penicillin G / Q000096:biosynthesis; D046915:Penicillin-Binding Proteins; D010407:Penicillium / Q000201:enzymology*; D010408:Penicillium chrysogenum / Q000201:enzymology*
D046915:Penicillin-Binding Proteins; D000105:Acetyl Coenzyme A; C037816:phenylacetyl-coenzyme A; D010397:Penicillanic Acid; D000217:Acyltransferases; C018756:acyl-CoA-6-aminopenicillanic acid acyltransferase; D010400:Penicillin G; C004408:aminopenicillanic acid; D004229:Dithiothreitol