title: Glutamate-119 of the large alpha-subunit is the catalytic base in the hydration of 2-trans-enoyl-coenzyme A catalyzed by the multienzyme complex of fatty acid oxidation from Escherichia coli.
abstract: Glu139 of the large alpha-subunit of the multienzyme complex of fatty acid oxidation from Escherichia coli was identified as the catalytic residue of enoyl-CoA hydratase [Yang, S.-Y., He, X.-Y., & Schulz, H. (1995) Biochemistry 34, 6441-6447]. To determine whether any of the other conserved protic residues is directly involved in the hydratase catalysis, the multienzyme complexes with either an alpha/Asp69 --> Asn or an alpha/Glu119 --> Gln mutation were overproduced and characterized. The catalytic properties of 3-ketoacyl-CoA thiolase and l-3-hydroxyacyl-CoA dehydrogenase of the mutant complexes were almost unaffected. The amidation of Asp69 and Glu119 caused a 7.6- and 88-fold decrease, respectively, in the kcat of enoyl-CoA hydratase without a significant change in the Km value of the hydratase as well as a 5.9- and 62-fold increase, respectively, in the Km of Delta3-cis-Delta2-trans-enoyl-CoA isomerase with a very small decrease in the kcat of the latter enzyme. The data suggest that the carboxyl group of Glu119 is particularly important to the catalytic activity of enoyl-CoA hydratase. Furthermore, the wild-type hydratase shows a bell-shaped pH dependence of the kcat/Km with pKa values of 5.9 and 9.2, whereas the Glu119 --> Gln mutant hydratase has only a single pKa of 9.5. A simple explanation for these observations is that a deprotonated Glu119 and a protonated Glu139 are required for the high kcat of the enoyl-CoA hydratase. The results of site-directed mutagenesis studies, together with the structural information about the spatial arrangement of two conserved glutamate residues of rat liver enoyl-CoA hydratase [Engel, C. K., Mathieu, M., Zeelen, J. P., Hiltunen, J. K., and Wierenga, R. K. (1996) EMBO J. 15, 5135-5145] to which Glu119 and Glu139 of the large alpha-subunit correspond, lead to the conclusion that the gamma-carboxyl group of Glu119 serves as the second general acid-base functional group in catalyzing the hydration of 2-trans-enoyl-CoA.
authors.affiliation: Department of Pharmacology, New York State Institute for Basic Research in Developmental Disabilities, Staten Island, New York 10314, USA.
authors.forename: X Y
authors.initials: XY
authors.lastname: He
authors.forename: S Y
authors.initials: SY
authors.lastname: Yang
mesh_terms: D000214:Acyl Coenzyme A / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D003065:Coenzyme A / Q000378:metabolism*; D004746:Enoyl-CoA Hydratase / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D005227:Fatty Acids / Q000378:metabolism*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D051381:Rats; D016415:Sequence Alignment
chemical_list: D000214:Acyl Coenzyme A; D005227:Fatty Acids; D009097:Multienzyme Complexes; C010701:crotonyl-coenzyme A; D004746:Enoyl-CoA Hydratase; D003065:Coenzyme A