The reductive half-reaction in acyl-CoA oxidase from Candida tropicalis: interaction with acyl-CoA analogues and an unusual thioesterase activity.
A series of acyl-CoA analogues has been used to probe the substrate binding site and reductive half-reaction of acyl-CoA oxidase from the alkane utilizing yeast Candida tropicalis. Alkyl-SCoA thioethers, from octyl- to hexadecyl-SCoA, bind to the oxidase with progressively larger spectral perturbation of the flavin chromophore and with an incremental binding energy of about 260 cal/methylene group. The hydrocarbon binding subsite for acyl-CoA oxidase appears extensive and only weakly hydrophobic. CoA binding per se appears to contribute about 2.8 kcal to the observed binding energy. A number of acyl-CoA analogues such as 3-thia-acyl-, 3-oxa-acyl-, trans-3-enoyl-, and 3-keto-acyl-CoA derivatives form charge transfer complexes with the oxidase, but these long wavelength bands are both less pronounced and much less stable than those encountered with the acyl-CoA dehydrogenases. This instability reflects an intrinsic thioesterase activity of the oxidase which is observed with those ligands forming enolate to oxidized flavin charge-transfer complexes, but not with normal substrates such as palmitoyl-CoA. Chemical precedent suggests that these enzyme-bound enolates eliminate CoA via a ketene intermediate. The differences in behavior between acyl-CoA oxidase and dehydrogenase toward the ligands used in this work are discussed in terms of the need to exclude oxygen from productive encounters with substrate-reduced dehydrogenase.
Wang
R
R
Department of Chemistry and Biochemistry, University of Delaware, Newark 19716.
Thorpe
C
C
D000214:Acyl Coenzyme A / Q000378:metabolism*; D042962:Acyl-CoA Oxidase; D001665:Binding Sites; D002175:Candida / Q000201:enzymology*; D007700:Kinetics; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism*; D011485:Protein Binding; D013053:Spectrophotometry; D013379:Substrate Specificity
D000214:Acyl Coenzyme A; D010088:Oxidoreductases; D042962:Acyl-CoA Oxidase