Histidine-450 is the catalytic residue of L-3-hydroxyacyl coenzyme a dehydrogenase associated with the large alpha-subunit of the multienzyme complex of fatty acid oxidation from Escherichia coli

作者：He XY; Yang SY

来源：Biochemistry, 1996, 35(29): 9625-9630.

DOI：10.1021/bi960374y

Multienzyme complexes of fatty acid oxidation fr om Escherichia coli with Gln or Ala substituting for His(450) or with Ala in place of Gly(322) in the large alpha-subunit have been purified and characterized. The alpha/Gly(322)-->Ala mutation did not significantly affect the catalytic efficiencies (k(cat)/K-m) of different component enzymes except for a 6.1-fold decrease in the k(cat)/K-m of L-3-hydroxyacyl-CoA dehydrogenase and a 10-fold increase in the K-m 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 k(cat)/K-m of the E. coli dehydrogenase suggested the catalytic involvement of an amino acid residue with a pK(a) of 6, which is presumably a histidine residue as proposed previously on the basis of chemical modifications. Since His(450) 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, His(450) was replaced by either Gin or Ala. The catalytic properties of 3-ketoacyl-CoA thiolase, enoyl-CoA hydratase, and Delta(3)-cis-Delta(2)-trans-enoyl-CoA isomerase of the alpha/His(450), Gln mutant complex were virtually unchanged except for a small decrease in the k(cat) 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 k(cat) and a 6-fold increase in the K-m for NADH. The alpha/His(450)-->Ala mutant complex showed similar catalytic behaviors. Taken together, several lines of evidence lead to the conclusion that His(450) is the catalytic residue of L-3-hydroxyacyl-CoA dehydrogenase of the E. coli multifunctional fatty acid oxidation protein.

• 出版日期1996-7-23