β-Cyclopiazonate oxidocyclase from Penicillium cyclopium. II. Studies on electron acceptors, inhibitors, enzyme kinetics, amino acid composition, flavin prosthetic group and other properties

Abstract

1. 1. 2,6-Dichlorophenolindophenol, phenazine methosulphate and cytochrome c were effective electron acceptors in the dehydrogenation reaction catalysed by the β-cyclopiazonate oxidocyclases. Oxygen was an effective terminal electron acceptor, but only in the presence of phenazine methosulphate as an intermediate electron carrier. Other electron acceptors and carriers were studied as was the effect of various metal ions and possible inhibitors. Most bivalent metal ions were found to inhibit the enzymes. Inhibition of the dehydrogenation and cyclization reactions by 2,4-dinitro-1-fluorobenzene and l-1-tosylamide-2-phenylethylchloromethyl ketone indicates the possible roles of amino groups and the histidine imidazole group in these reactions. Similar conclusions were made from studies of the effect of pH on the reactions. Mechanisms involving the abovementioned groups are proposed for the dehydrogenation as well as the cyclization reaction. 2. 2. The thermodynamic properties of the isoenzymes were found to be similar. With respect to the dehydrogenation reaction, the following values were found: activation energy ( E a approx. 7.2 kcal· mole −1); enthalpy change of activation ( ΔH ∗ approx. 6.6 kcal·mole −1); entropy change of activation ( ΔS ∗ approx. −42 cal·mole −1. degree −1); and free energy change of activation ( ΔG ∗ approx. 19.3 kcal·mole −1). The five isoenzymes were also similar with respect to their response towards inhibitors and electron acceptors; K m values for 2,6-dichlorophenolindophenol ( K m approx. 1.6 μM, isoenzyme II somewhat higher) and catalytic constants ( k 2 approx. 0.048 sec −1, isoenzyme II somewhat lower). 3. 3. The isoenzymes of β-cyclopiazonate oxidocyclase differed in the order of the dehydrogenation reaction with respect to β-cyclopiazonic acid, the half periods of the dehydrogenation reaction, the Michaelis constants for β-cyclopiazonic acid (2.1–14 μM), amino acid composition as far as the ratio of basic to acidic amino acids are concerned, and ammonia and carbohydrate content. 4. 4. All the isoenzymes contained one covalently linked molecule of flavin per molecule of enzyme. Absorption spectra of the isoenzymes showed characteristic peaks at 276, 366 and 450 nm and a shoulder at 290 nm. Their fluorescence emission spectra showed a peak at about 527 nm and the flavin residue (most probably FAD), released by proteolytic digestion with pronase showed a fluorescence peak at 518 nm similar to that of FMN and FAD.