Mechanism of barbiturate inhibition on purified flavoenzymes

Abstract

Summary The flavoenzymes D -amino acid oxidase (EC 1.4.3.3), L -amino acid oxidase (EC 1.4.3.2), aldehyde oxidase (EC 1.2.3.1), NAD(P)H dehydrogenases (EC 1.6.5.2) and D -aspartate oxidase (EC 1.4.3.1) are inhibited by phenobarbital with a mechanism of competition with the substrate; the flavoenzymes glucose oxidase (EC 1.1.3.4), lipoic dehydrogenase (EC 1.6.4.3) and NADPH-glutathione reductase (EC 1.6.4.2) are not inhibited. Of the non-flavoenzymes examined, fumarase (EC 4.2.1.2), catalase (EC 1.11.1.6) and ribonuclease (EC 2.7.7.16) are not inhibited, while alcohol dehydrogenase (EC 1.1.1.1), lactate dehydrogenase (EC 1.1.1.27) and aspartate aminotransferase (EC 2.6.1.1) are inhibited at relatively higher concentrations of barbiturate. The flavin spectra of D -amino acid oxidase and D -aspartate oxidase are sensibly modified by phenobarbital. A three-fold increase in the flavin fluorescence of D -amino acid oxidase is also brought about by phenobarbital which does not modify the conformation of the protein as measured by the bromothymol blue method. The rate and extent of formation of the flavin semiquinone during the catalytic cycle of D -amino acid oxidase is strongly inhibited by phenobarbital. Higher concentrations of phenobarbital produce modifications in the absorption and fluorescence properties of solutions of flavin nucleotides. These data are taken to suggest an interaction of phenobarbital with enzyme-bound flavins, although a binding with the substrate site may also occur in some cases.