Importance of the γ-Carboxyl Group of Glutamate-462 of the Large α-Subunit for the Catalytic Function and the Stability of the Multienzyme Complex of Fatty Acid Oxidation from Escherichia coli
- 1 January 1997
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 36 (1) , 261-268
- https://doi.org/10.1021/bi961841e
Abstract
His450 of the large α-subunit of the multienzyme complex of fatty acid oxidation from Escherichia coli was recently identified as an essential catalytic residue of l-3-hydroxyacyl-CoA dehydrogenase [He, X.-Y., & Yang, S.-Y. (1996) Biochemistry 35, 9625−9630]. To explore the roles of acidic residues in the dehydrogenase catalysis, every conserved acidic residue in the dehydrogenase functional domain except for those in the NAD-binding motif was replaced with alanine. The resulting mutant complexes were overproduced and characterized. Their component enzymes other than the dehydrogenase were affected very slightly. Removal of the β-carboxyl group of Asp524 and Asp542 caused only a 3- and 4-fold, respectively, decrease in the catalytic efficiency of the dehydrogenase, thereby showing that their involvement in the dehydrogenase catalysis was limited. In contrast, the α/Glu462 → Ala mutant complex showed a greater than 160-fold reduction in the kcat of the dehydrogenase in the forward direction without a significant change of the Km for the substrate. The catalytic properties of the α/Glu462 → Gln mutant complex were found to be similar to those of the α/Glu462 → Ala mutant complex except that the kcat of the dehydrogenase in the backward direction was about 4-fold lower and the Km for the substrate of the thiolase was 6-fold higher. It is concluded that the negative charge of the γ-carboxyl group of Glu462, but not its ability to form a hydrogen bond, is critical for its interaction with His450, thereby assisting in the catalysis of the dehydrogenase. The pKa of His450 in the E·NADH binary complex was virtually unchanged by the replacement of Glu462 with Ala or Gln. It seems that the binding of substrate is necessary for forming a strong interaction between His450 and Glu462 with the result that the electroneutrality in the active site is maintained and the activation energy of the reaction is lowered. Additionally, the negative charge of Glu462 increases the thermostability of the multienzyme complex.Keywords
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