alexa Histidine-450 Is the Catalytic Residue of l-3-Hydroxyacyl Coenzyme A Dehydrogenase Associated with the Large α-Subunit of the Multienzyme Complex of Fatty Acid Oxidation from Escherichia coli
Microbiology

Microbiology

Journal of Chemical Biology & Therapeutics

Author(s): XueYing, SongYu Yang

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Multienzyme complexes of fatty acid oxidation from Escherichia coli with Gln or Ala substituting for His450 or with Ala in place of Gly322 in the large α-subunit have been purified and characterized. The α/Gly322 → Ala mutation did not significantly affect the catalytic efficiencies (kcat/Km) of different component enzymes except for a 6.1-fold decrease in the kcat/Km of l-3-hydroxyacyl-CoA dehydrogenase and a 10-fold increase in the Km for NADH. This observation confirms the prediction [Yang, X.-Y. H., Schulz, H., Elzinga, M., & Yang, S.-Y. (1991) Biochemistry 30, 6788−6795] that the E. coli dehydrogenase has an NAD-binding site at its amino-terminal domain and structurally resembles the pig heart dehydrogenase. The pH dependence of the kcat/Km of the E. coli dehydrogenase suggested the catalytic involvement of an amino acid residue with a pKa of 6, which is presumably a histidine residue as proposed previously on the basis of chemical modifications. Since His450 of the E. coli multifunctional protein is the only histidine conserved in all known l-3-hydroxyacyl-CoA dehydrogenases, and since its counterpart in pig heart enzyme appeared to be close to the 3-keto group of the fatty acyl moiety of the substrate, His450 was replaced by either Gln or Ala. The catalytic properties of 3-ketoacyl-CoA thiolase, enoyl-CoA hydratase, and Δ3-cis-Δ2-trans-enoyl-CoA isomerase of the α/His450 → Gln mutant complex were virtually unchanged except for a small decrease in the kcat values of the latter two enzymes. In contrast, the dehydrogenase of this mutant complex was almost inactive due to a greater than 3000-fold decrease in its kcat and a 6-fold increase in the Km for NADH. The α/His450 → Ala mutant complex showed similar catalytic behaviors. Taken together, several lines of evidence lead to the conclusion that His450 is the catalytic residue of l-3-hydroxyacyl-CoA dehydrogenase of the E. coli multifunctional fatty acid oxidation protein.

This article was published in Biochemistry and referenced in Journal of Chemical Biology & Therapeutics

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