Flavin substrate specificity of the vitamin B2-aldehyde-forming enzyme from Schizophyllum commune.

Vitamin B2-aldehyde-forming enzyme from Schizophyllum commune catalyzes oxidation of the 5'-hydroxymethyl of riboflavin to the formyl group. We have monitored enzyme activity by spectrophotometrically measuring the reduction of 2,6-dichlorophenol-indolphenol as electron acceptor to assess 35 riboflavin analogs as potential substrates or competitive inhibitors with the purpose of delimiting structural requirements of the substrate binding site. Analogs with side chains of two- to six-carbon length modified by deletion of secondary hydroxyls or by changes in their epimeric configuration are not oxidized. The omega-hydroxyalkyl-flavins (n = 2-6) are competitive inhibitors (Ki = 7-16 microM) of riboflavin oxidation, as are some analogs with L-secondary hydroxyls in the side chain. Analogs with bulky substituents on the isoalloxazine ring are also not substrates. The enzyme does not significantly bind flavins with an 8 alpha-N-imidazole; diethylamino, methylethylamino, dimethylamino, ethylamino, or ethoxy groups at position 8; methyl at 6; and beta-hydroxyethylamino at position 2. Also the replacement of N with CH in 1-deazariboflavin disallows substrate reaction. Analogs with fluoro, chloro, methyl, amino, or methylamino at position 8; chloro at 7; methyl or carboxylmethyl at 3; thio at 2, and C replacing N at positions 3 or 5 are substrates with relative Vmax values ranging from 27 to 110% that of riboflavin. The Km values for the analogs oxidized are all found to be in the micromolar range (22-176 microM). Overall specificity of the enzyme for riboflavin is found to be rather narrow and sterically limited, which suggests that the vitamin is the natural substrate.

Hysteretic interaction of NADH and Mg2+ with mammalian NADH:CoQ reductase from beef heart.

Preincubation of submitochondrial particles (SMP) from beef heart in a reaction mixture containing low concentrations of Mg2+ induces a time lag in the NADH:oxidase activity. Preconditioning of the SMP by NADH, but not by NAD+, prevents the Mg2+-related time lag. The data obtained show that there exists a tight binding site for Mg2+ regulating the rate of electron transfer from NADH to the natural acceptor. The ability of Mg2+ to form a catalytically inactive complex with the enzyme is regulated by NADH.