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 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.
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Department of Pharmacology, New York State Institute for Basic Research in Developmental Disabilities, Staten Island 10314, USA.
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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
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