Functional role of the active site glutamate-368 in rat short chain acyl-CoA dehydrogenase.
The acyl-CoA dehydrogenases are a family of flavoenzymes with similar structure and function involved in the metabolism of fatty acids and branched chain amino acids. The degree of overlap in substrate specificity is narrow among these enzymes. The position of the catalytic glutamate, identified as Glu376 in porcine medium chain acyl-CoA dehydrogenase (MCAD), Glu254 in human isovaleryl-CoA dehydrogenase (IVD), and Glu261 in human long chain acyl-CoA dehydrogenase (LCAD), has been suggested to affect substrate chain length specificity. In this study, in vitro site-directed mutagenesis was used to investigate the effect of changing the position of the catalytic carboxylate on substrate specificity in short chain acyl-CoA dehydrogenase (SCAD). Glu368, the hypothetical active site catalytic residue of rat SCAD, was replaced with Asp, Gly, Gln, Arg, and Lys and the wild type and mutant SCADs were produced in Escherichia coli and purified. The recombinant wild type SCAD kcat/K(m) values for butyryl-hexanoyl-, and octanoyl-CoA were 220, 22, and 3.2 microM-1 min-1, respectively, while the Glu368Asp mutant gave kcat/K(m) of 81, 12, and 1.4 microM-1 min-1, respectively, for the same substrates. None of the other mutants exhibited enzyme activity. A Glu368Gly/Gly247Glu double mutant enzyme, which places the catalytic residue at a position homologous to that of LCAD, was also synthesized and purified. It showed kcat/K(m) of 9.3, 2.8, and 1.5 microM-1 min-1 with butyryl-, hexanoyl-, and octanoyl-CoA used as substrates, respectively. These results confirm the identity of Glu368 as the catalytic residue of rat SCAD and suggest that alteration of the position of the catalytic carboxylate can modify substrate specificity.
Battaile
K P
KP
Department of Medical Genetics, Mayo Clinic, Rochester, Minnesota 55905, USA.
Mohsen
A W
AW
Vockley
J
J
D042964:Acyl-CoA Dehydrogenase; D044942:Acyl-CoA Dehydrogenase, Long-Chain / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004926:Escherichia coli; D018698:Glutamic Acid; D006801:Humans; D008958:Models, Molecular; D011487:Protein Conformation; D051381:Rats; D013054:Spectrophotometry, Atomic
D018698:Glutamic Acid; D042964:Acyl-CoA Dehydrogenase; D044942:Acyl-CoA Dehydrogenase, Long-Chain