Functional Role of the Active Site Glutamate-368 in Rat Short Chain Acyl-CoA Dehydrogenase
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- 作者：Kevin P. Battaile ; Al-Walid A. Mohsen ; and Jerry Vockley
- 刊名：Biochemistry
- 出版年：1996
- 出版时间：December 3, 1996
- 年：1996
- 卷：35
- 期：48
- 页码：15356 - 15363
- 全文大小：378K
- 年卷期：v.35,no.48(December 3, 1996)
- ISSN：1520-4995
文摘
The acyl-CoA dehydrogenases are a family of flavoenzymes withsimilar structure and functioninvolved in the metabolism of fatty acids and branched chain aminoacids. The degree of overlap insubstrate specificity is narrow among these enzymes. The positionof the catalytic glutamate, identifiedas Glu376 in porcine medium chain acyl-CoA dehydrogenase (MCAD), Glu254in human isovaleryl-CoA dehydrogenase (IVD), and Glu261 in human long chain acyl-CoAdehydrogenase (LCAD), hasbeen suggested to affect substrate chain length specificity. Inthis study, in vitro site-directed mutagenesiswas used to investigate the effect of changing the position of thecatalytic carboxylate on substrate specificityin short chain acyl-CoA dehydrogenase (SCAD). Glu368, thehypothetical active site catalytic residue ofrat SCAD, was replaced with Asp, Gly, Gln, Arg, and Lys and the wildtype and mutant SCADs wereproduced in Escherichia coli and purified. Therecombinant wild type SCADkcat/Km values forbutyryl-,hexanoyl-, and octanoyl-CoA were 220, 22, and 3.2
M-1 min-1,respectively, while the Glu368Aspmutant gave kcat/Km of81, 12, and 1.4 M-1min-1, respectively, for the same substrates.None of theother mutants exhibited enzyme activity. A Glu368Gly/Gly247Gludouble mutant enzyme, which placesthe catalytic residue at a position homologous to that of LCAD, wasalso synthesized and purified. Itshowed kcat/Km of 9.3,2.8, and 1.5 M-1min-1 with butyryl-, hexanoyl-, andoctanoyl-CoA used assubstrates, respectively. These results confirm the identity ofGlu368 as the catalytic residue of rat SCADand suggest that alteration of the position of the catalyticcarboxylate can modify substrate specificity.
M-1 min-1,respectively, while the Glu368Aspmutant gave kcat/Km of81, 12, and 1.4 M-1min-1, respectively, for the same substrates.None of theother mutants exhibited enzyme activity. A Glu368Gly/Gly247Gludouble mutant enzyme, which placesthe catalytic residue at a position homologous to that of LCAD, wasalso synthesized and purified. Itshowed kcat/Km of 9.3,2.8, and 1.5 M-1min-1 with butyryl-, hexanoyl-, andoctanoyl-CoA used assubstrates, respectively. These results confirm the identity ofGlu368 as the catalytic residue of rat SCADand suggest that alteration of the position of the catalyticcarboxylate can modify substrate specificity.