PMID:7756275
| Citation |
Yang, SY, He, XY and Schulz, H (1995) Glutamate 139 of the large alpha-subunit is the catalytic base in the dehydration of both D- and L-3-hydroxyacyl-coenzyme A but not in the isomerization of delta 3, delta 2-enoyl-coenzyme A catalyzed by the multienzyme complex of fatty acid oxidation from Escherichia coli. Biochemistry 34:6441-7 |
|---|---|
| Abstract |
Multienzyme complexes of fatty acid oxidation from Escherichia coli with either an alpha/Glu139-->Gln or an alpha/Arg134-->Gln mutation in the large alpha-subunit have been overproduced and characterized. The catalytic properties of the five different component enzymes of the alpha/Arg134-->Gln mutant complex showed no significant changes as compared with those of the wild type complex. In contrast, the 3-hydroxyacyl-coenzyme A (CoA) epimerase activity of the alpha/Glu139-->Gln mutant complex was not detected, and this mutant complex has lost almost all of the enoyl-CoA hydratase activity due to a greater than 3000-fold decrease in the kcat of the enoyl-CoA hydratase without a significant change in the Km value. The catalytic properties of 3-ketoacyl-CoA thiolase and L-3-hydroxyacyl-CoA dehydrogenase were virtually unaffected by the mutation. Together, these observations lead to the conclusion that the gamma-carboxylic group of Glu139 functions as a catalytic base in the dehydration of both D- and L-3-hydroxyacyl-CoA. These findings also support a dehydration/hydration mechanism for 3-hydroxyacyl-CoA epimerase but do not agree with an epimerase activity independent of enoyl-CoA hydratase as proposed for the glyoxysomal tetrafunctional protein [Preisig-Müller, R., Gühnemann-Schäfer, K., & Kindl, H. (1994) J. Biol. Chem. 269, 20475-20481]. Since this mutation caused the kcat of delta 3-cis-delta 2-trans-enoyl-CoA isomerase to decrease by only 60%, even though the Km value was significantly increased, it seems that Glu139 of the E. coli multifunctional protein does not function as a catalytic residue in the isomerization reaction.(ABSTRACT TRUNCATED AT 250 WORDS) |
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| Keywords |
3-Hydroxyacyl CoA Dehydrogenases/chemistry; 3-Hydroxyacyl CoA Dehydrogenases/metabolism; Acetyl-CoA C-Acyltransferase/chemistry; Acetyl-CoA C-Acyltransferase/metabolism; Acyl Coenzyme A/metabolism; Amino Acid Sequence; Animals; Base Sequence; Binding Sites; Carbon-Carbon Double Bond Isomerases; Catalysis; DNA Primers/chemistry; Enoyl-CoA Hydratase/chemistry; Enoyl-CoA Hydratase/metabolism; Escherichia coli/enzymology; Glutamates; Isomerases/chemistry; Isomerases/metabolism; Molecular Sequence Data; Racemases and Epimerases/chemistry; Racemases and Epimerases/metabolism; Rats; Sequence Alignment; Sequence Homology, Amino Acid |
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