| dc.description.abstract | The two first enzymes in the pathway for the metabolism of mandelic acid by Aspergillus niger, namely D(-)-mandelate oxidase and L(+)-mandelate oxidase, which respectively catalyze the oxidation of the two enantiomers of mandelic acid to benzoylformic acid, have been studied.
L(+)-mandelate oxidase has been purified about 35-fold by treatment of the crude extracts with protamine sulphate, adsorption onto and elution from DEAE-cellulose, and negative adsorption on alumina gel and tricalcium phosphate gel. The enzyme is highly labile; approximate half-life is 6 hours at pH 7.0 and at 0–5°C. It is maximally active at pH 7.0 and at 35°C. Oxygen and 2,6-dichlorophenolindophenol function as the electron acceptors; in the former case, the reaction is accompanied by the formation of hydrogen peroxide in stoichiometric quantities. The purified enzyme is the apoenzyme of L(+)-mandelate oxidase and shows an absolute requirement for flavin nucleotides. FMN is about 30% as effective as FAD. The Km for L(+)-mandelate is 33 ?M and for FAD is 1.7 ?M. The enzyme has an approximate molecular weight of 115,000. The energy of activation for the oxidase reaction calculated from the Arrhenius plot is 9984 calories per mole of L-mandelate.
D(-)-mandelate oxidase of Aspergillus niger is a particulate enzyme and is comparatively less stable than L-mandelate oxidase. All attempts to solubilize the enzyme have been a failure; the properties of the enzyme have been studied using the particulate fraction. The enzyme shows a fairly sharp optimum at pH 7.6 and is maximally active at 35°C. Electrons removed from the substrate seem to be transferred to oxygen through the cytochrome system. 2,6-dichlorophenolindophenol or ferricyanide are not effective as electron acceptors.
Both the enzymes are susceptible to inhibition by iron-chelating and copper-chelating agents, thiol-binding agents, and heavy metal ions. Neither enzyme is capable of reducing pyridine nucleotides or exogenously added cytochrome c. Experiments with substrate analogues suggest that the structural requirements for both the enzymes on the substrate are:
(a) free carboxyl group,
(b) a suitably substituted benzene ring on the 2-carbon atom, and
(c) a 2-hydroxyl group, all arranged in the appropriate configuration.
Experiments on induction and repression suggest that the first three enzymes in the mandelate pathway of Aspergillus niger, namely D(-)-mandelate oxidase, L(+)-mandelate oxidase, and benzoylformate decarboxylase, are controlled by block-induction and probably form part of the control system of a regulon, which is inducible by D(-)-, L(+)- and DL-mandelates, benzoylformate, benzaldehyde, and ring-substituted mandelates, and repressible by further products of mandelate degradation, namely benzoate, 4-hydroxybenzoate, and 3,4-dihydroxybenzoate. | |
