The overall objective is to elucidate the molecular basis of the inherited metabolic disorders of fatty acid metabolism due to a deficiency of short- (SCAD), medium- (MCAD) and long chain acyl-CoA dehydrogenases (LCAD). These disorders are important in the differential diagnosis of children with episodic acidosis, hypoglycemia or myopathy. SCAD, MCAD, and LCAD catalyze the first reaction of the Beta oxidation cycles in the fatty acid catabolism. They are all mitochondrial flavoenzymes. These three enzymes and two other acyl-CoA dehydrogenases, that are involved in the branched chain amino acid metabolism, share many similarities in the molecular weight, subunit structure, active site, and reaction mechanisms. They differ in the length and steric configuration of the acyl-CoA substrate, however. These considerations led us to hypothesize that these enzymes belong to a family (the acyl-CoA dehydrogenase family) and share a common ancestor, but acquired distinctive structural diversity and substrate specificity in the course of evolution. This hypothesis must be tested, however, by the determination of nucleotide sequences of cDNA's. Individual experiments proposed are: 1) purification and characterization of human SCAD, MCAD and LCAD; 2) study of residual activities in the cells from patients with a deficiency of SCAD or MCAD; 3) biosynthesis of variant SCAD and LCAD in cultured fibroblasts from patients with a deficiency of the respective enzyme; 4) amino acid sequencing of the peptide containing the essential cysteine residue that is located in the active site, and other tryptic peptides; 5) molecular cloning of cDNA's encoding SCAD, MCAD, and LCAD; 6) chromosome mapping of SCAD, MCAD, and LCAD; 7) large scale preparation of MCAD using cloned cDNA and an expression vector for the study of the three dimensional structures by X-ray crystallography 8) study of single base substitution(s) in variant MCAD using ribonuclease A; 9) identification of point mutation in variant MCAD gene; and 10) molecular study of mutant human SCAD and LCAD genes.
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