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.
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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Department of Pharmacology, New York State Institute for Basic Research in Developmental Disabilities, Staten Island 10314, USA.
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Schulz
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D015094:3-Hydroxyacyl CoA Dehydrogenases / Q000737:chemistry / Q000378:metabolism*; D000102:Acetyl-CoA C-Acyltransferase / Q000737:chemistry / Q000378:metabolism*; D000214:Acyl Coenzyme A / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D019748:Carbon-Carbon Double Bond Isomerases; D002384:Catalysis; D017931:DNA Primers / Q000737:chemistry; D004746:Enoyl-CoA Hydratase / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D005971:Glutamates; D066298:In Vitro Techniques; D007535:Isomerases / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D019998:Racemases and Epimerases / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D000214:Acyl Coenzyme A; D017931:DNA Primers; D005971:Glutamates; C038784:fatty acid oxidation complex; 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