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 bicycli sation of delta-(L-alpha -aminoadipoyl)-L-cysteinyl-D-valine into isopenici llin N, the precursor of all other penicillins, cephalosporins and cephamyc ins, The IPNS reaction can be followed directly and continuously by a new a ssay which monitors the absorbance increase at 235 nm characteristic of pen icillin nucleus formation. Using this assay, the effects of influential fac tors affecting the in vitro IPNS enzymatic reaction were investigated. Even under optimal conditions, enzyme inactivation occurred during catalysis, I ron(II) depletion and product inhibition were not the cause of this phenome non, the addition of antioxidants or reducing agents failed to slow down in activation 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, Neverthe less, the steady-state kinetic parameters for the IPNS reaction mere determ ined. (C) 2000 Federation of European Biochemical Societies. Published by E lsevier Science B.V. All rights reserved.