Studies of isopenicillin N synthase enzymatic properties using a continuous spectrophotometric assay.
Isopenicillin N synthase (IPNS) from Aspergillus nidulans is a no-heme iron(II)-dependent oxygenase which catalyses, in a single reaction, the bicyclisation of delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine into isopenicillin N, the precursor of all other penicillins, cephalosporins and cephamycins. The IPNS reaction can be followed directly and continuously by a new assay which monitors the absorbance increase at 235 nm characteristic of penicillin nucleus formation. Using this assay, the effects of influential factors affecting the in vitro IPNS enzymatic reaction were investigated. Even under optimal conditions, enzyme inactivation occurred during catalysis. Iron(II) depletion and product inhibition were not the cause of this phenomenon, the addition of antioxidants or reducing agents failed to slow down inactivation or reactivate the enzyme. Therefore, this phenomenon appears to be irreversible and is attributed to oxidative damage caused to the enzyme by reactive oxygen species generated in solution during catalysis. Nevertheless, the steady-state kinetic parameters for the IPNS reaction were determined.
Dubus
A
A
Laboratoire d'Enzymologie, CIP, Institut di Chimie B6, University of Liège, Belgium. a.dubus@ulg.ac.be
Sami
M
M
Brown
T J
TJ
Schofield
C J
CJ
Baldwin
J E
JE
Frère
J M
JM
D000975:Antioxidants / Q000494:pharmacology; D001205:Ascorbic Acid / Q000494:pharmacology; D001233:Aspergillus nidulans / Q000201:enzymology; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D004229:Dithiothreitol / Q000494:pharmacology; D004793:Enzyme Reactivators / Q000494:pharmacology; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D010088:Oxidoreductases / Q000378:metabolism*; D010406:Penicillins / Q000378:metabolism; D019163:Reducing Agents / Q000494:pharmacology; D013056:Spectrophotometry, Ultraviolet
D000975:Antioxidants; D004793:Enzyme Reactivators; D010406:Penicillins; D019163:Reducing Agents; D007501:Iron; D010088:Oxidoreductases; C038370:isopenicillin N synthetase; C006414:penicillin N; D001205:Ascorbic Acid; D004229:Dithiothreitol