flavin-adenine-dinucleotide and isovaleric-acid

Medium-chain acyl-coenzyme A (CoA) dehydrogenase (MCADH; EC 1.3.99.3) deficiency (MCD) is an inborn error of beta-oxidation. We measured 3H2O formed by the dehydrogenation of [2,3-3H]acyl-CoAs in a 3H-release assay. Short-chain acyl-CoA dehydrogenase (SCADH; EC 1.3.99.2), MCADH, and isovaleryl-CoA dehydrogenase (IVDH; EC 1.3.99.10) activities were assayed with 100 microM [2,3-3H]butyryl-, -octanoyl-, and -isovaleryl-CoAs, respectively, in fibroblasts cultured from normal controls and MCD patients. Without the artificial electron acceptor phenazine methosulfate (PMS), MCADH activity in fibroblast mitochondrial sonic supernatants (MS) was 54% of control in two MCD cell lines (P less than 0.05). Addition of 10 mM PMS raised control acyl-CoA dehydrogenase activities 16-fold and revealed MCADH and SCADH activities to be 5 (P less than 0.01) and 73% (P greater than 0.1) of control, respectively. Thus, the catalytic defect in MCD involves substrate binding and/or dehydrogenation by MCADH and not the subsequent reoxidation of reduced MCADH by electron acceptors. 20 microM flavin adenine dinucleotide (FAD) did not stimulate MCD MCADH activity in either the 3H-release or electron-transfer(ring) flavoprotein-linked dye-reduction assays. Mixing experiments revealed no MCADH inhibitor in MCD MS; IVDH activities were identical in both control and MCD MS. In postmortem liver MS from another MCD patient, 3H2O formation from [2,3-3H]octanoyl-CoA was 15% of control. When 3H2O formation was assayed with 200 microM [2,3-3H]acyl-CoAs, 15 mM PMS, and 20 microM FAD in fibroblast sonic supernatants from seven MCD cell lines, SCADH, MCADH, and IVDH activities were 72-112% (P greater than 0.1), 4-9% (P less than 0.01), and 86-135% (P greater than 0.1) of control, respectively, revealing no significant biochemical heterogeneity among these patients.

Topics: Acyl Coenzyme A; Acyl-CoA Dehydrogenases; Adult; Fibroblasts; Flavin-Adenine Dinucleotide; Hemiterpenes; Humans; Infant; Isovaleryl-CoA Dehydrogenase; Lipid Metabolism, Inborn Errors; Male; Methylphenazonium Methosulfate; Mitochondria, Liver; Oxidoreductases; Oxidoreductases Acting on CH-CH Group Donors; Pentanoic Acids; Subcellular Fractions

Conversion of leucine to isovaleric acid by Clostridium bifermentans is achieved by the action of at least two enzymes. One is a transaminase producing alpha-ketoisocaproic acid, which was purified 30-fold from osmotic lysates of late-exponential phase cells by repeated chromatography on DEAE-Sepharose C16B and Sephacryl S300: this represented a 147-fold purification of activity found in sonically disrupted cells. This enzyme had an apparent molecular weight of approximately 190000 and was composed of six identically sized sub-units (molecular weight 31000 +/- 1000). Transamination required pyridoxal phosphate and pyruvate and was optimal at pH 8.6; the apparent Km for leucine was 7.0 mM. Activity was totally inhibited by 1 mM-p-chloromercuribenzoate and partially inhibited by other thiol reagents. The second enzyme decarboxylated alpha-ketoisocaproic acid to form isovaleric acid and was also partially purified by chromatography on DEAE-Sepharose C16B and Sephacryl S300. It has an apparent molecular weight of 240000 and required FAD and coenzyme A for activity; the Km for alpha-ketoisocaproic acid was 4.2 mM and activity was optimal around pH 8.0. This enzyme was a flavoprotein with absorption maxima at 280, 320 and 400 nm, and a fluorescent maximum at 500 nm. The prosthetic group, FAD, dissociated from the protein during purification resulting in an inactive apoenzyme which was only partially re-activated by FAD. Activity was completely inhibited by several thiol reagents tested at 1 mM.

Topics: Carboxy-Lyases; Chromatography, Gel; Chromatography, Ion Exchange; Clostridium; Deamination; Flavin-Adenine Dinucleotide; Hemiterpenes; Hydrogen-Ion Concentration; Keto Acids; Leucine; Molecular Weight; Pentanoic Acids; Transaminases; Valerates