Short chain acyl-CoA dehydrogenase (SCAD) is a homotetrameric flavoenzyme that catalyzes the first intramitochondrial step in the beta-oxidation of
fatty acids. Two polymorphisms in the coding region of the SCAD gene, 511C>T (R147W) and 625G>A (G185S), have been shown to be associated with an increased level of
ethylmalonic acid excretion in urine, a clinical characteristic of
SCAD deficiency. To characterize the biochemical consequences of these variations, in vitro site-directed mutagenesis and prokaryotic expression were used to produce the corresponding SCAD variant
proteins. Both variant
proteins were unstable when produced in Escherichia coli, but could be rescued and subsequently purified by coexpressing them with the bacterial
chaperonin GroEL/ES. The k(cat)/K(m) values of the green wild-type, R147W, and G185S SCAD
enzymes coexpressed with GroEL/ES were 33, 30, and 10 microM(-)(1) s(-)(1), respectively. There were minimal differences in the kinetic parameters measured for the green, degreened, and wild-type
enzymes coexpressed with GroEL/ES, and the R147W variant when
butyryl-CoA was used as a substrate. The catalytic efficiency of the G185S variant
enzyme, however, was reduced compared to that of the wild-type
enzyme. The thermal and
guanidine HCl stability of the purified
enzymes as determined by fluorescence, far-UV CD spectroscopy, and incubation-induced rest activity showed the following order of relative stability: wild-type
enzyme > R147W > G185S. Near-UV CD spectroscopy indicated that these impairments are caused by decreased flexibility in the tertiary conformation of the two mutant
enzymes. The common SCAD polymorphisms may lead to clinically relevant alterations in
enzyme function.