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

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.