Importance of the gamma-carboxyl group of glutamate-462 of the large alpha-subunit for the catalytic function and the stability of the multienzyme complex of fatty acid oxidation from Escherichia coli.
His450 of the large alpha-subunit of the multienzyme complex of fatty acid oxidation from Escherichia coli was recently identified as an essential catalytic residue of L-3-hydroxyacyl-CoA dehydrogenase [He, X-Y., & Yang, S.-Y. (1996) Biochemistry 35, 9625-9630]. To explore the roles of acidic residues in the dehydrogenase catalysis, every conserved acidic residue in the dehydrogenase functional domain except for those in the NAD-binding motif was replaced with alanine. The resulting mutant complexes were overproduced and characterized. Their component enzymes other than the dehydrogenase were affected very slightly. Removal of the beta-carboxyl group of Asp524 and Asp542 caused only a 3- and 4-fold, respectively, decrease in the catalytic efficiency of the dehydrogenase, thereby showing that their involvement in the dehydrogenase catalysis was limited. In contrast, the alpha/Glu462-->Ala mutant complex showed a greater than 160-fold reduction in the kcat of the dehydrogenase in the forward direction without a significant change of the k(m) for the substrate. The catalytic properties of the alpha/Glu462-->Gln mutant complex were found to be similar to those of the alpha/Glu462-->Ala mutant complex except that the kcat of the dehydrogenase in the backward direction was about 4-fold lower and the Km for the substrate of the thiolase was 6-fold higher. It is concluded that the negative charge of the gamma-carboxyl group of Glu462, but not its ability to form a hydrogen bond, is critical for its interaction with His450, thereby assisting in the catalysis of the dehydrogenase. The pKa of His450 in the E.NADH binary complex was virtually unchanged by the replacement of Glu462 with Ala or Gln. It seems that the binding of substrate is necessary for forming a strong interaction between His450 and Glu462 with the result that the electroneutrality in the active site is maintained and the activation energy of the reaction is lowered. Additionally, the negative charge of Glu462 increases the thermostability of the multienzyme complex.
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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; D001665:Binding Sites / Q000235:genetics; D019748:Carbon-Carbon Double Bond Isomerases; D017931:DNA Primers; D004746:Enoyl-CoA Hydratase / Q000737:chemistry* / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D018698:Glutamic Acid / Q000737:chemistry / Q000378:metabolism; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007535:Isomerases / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D011487:Protein Conformation; D011489:Protein Denaturation; D019998:Racemases and Epimerases / Q000737:chemistry* / Q000378:metabolism; D013696:Temperature
D017931:DNA Primers; C038784:fatty acid oxidation complex; D018698:Glutamic Acid; D006639:Histidine; 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