title: 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.
abstract: 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 alpha-subunit have been purified and characterized. The alpha/Gly322-->Ala mutation did not significantly affect the catalytic efficiencies (kcat/k(m)) of different component enzymes except for a 6.1-fold decrease in the kcat/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 kcat/k(m) 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 delta 3-cis-delta 2-trans-enoyl-CoA isomerase of the alpha/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 k(m) for NADH. The alpha/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.
authors: Department of Pharmacology, New York State Institute for Basic Research in Developmental Disabilities, Staten Island 10314, USA.
authors: X Y
authors: XY
authors: He
authors: S Y
authors: SY
authors: Yang
mesh_terms: D015094:3-Hydroxyacyl CoA Dehydrogenases / Q000737:chemistry* / Q000378:metabolism*; D000102:Acetyl-CoA C-Acyltransferase / Q000737:chemistry / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D019748:Carbon-Carbon Double Bond Isomerases; D004591:Electrophoresis, Polyacrylamide Gel; D004746:Enoyl-CoA Hydratase / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D006639:Histidine / Q000378:metabolism*; D007535:Isomerases / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D019998:Racemases and Epimerases / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017421:Sequence Analysis
chemical_list: D010446:Peptide Fragments; D011994:Recombinant Proteins; C038784:fatty acid oxidation complex; D006639:Histidine; D000244:Adenosine Diphosphate; D015094:3-Hydroxyacyl CoA Dehydrogenases; D000102:Acetyl-CoA C-Acyltransferase; D004746:Enoyl-CoA Hydratase; D007535:Isomerases; D019998:Racemases and Epimerases; D019748:Carbon-Carbon Double Bond Isomerases