Inhibition of acyl-CoA oxidase by phenol and its implication in measurement of the enzyme activity via the peroxidase-coupled assay system.
Yeast (Candida tropicalis) acyl-CoA oxidase catalyzes the oxidation of a variety of acyl-CoA substrates to their corresponding alpha-beta enoyl-CoA products, with concomitant reduction of the buffer-dissolved O2 to H2O2. By utilizing indolepropionyl-CoA as a chromogenic substrate, we could measure the enzyme activity either directly by monitoring formation of the reaction product indoleacryloyl-CoA (lambda(max) = 367 nm) or indirectly by measuring the formation of H2O2 via the oxidative-coupled assay system, involving 4-aminoantipyrine, phenol, and horseradish peroxidase. We compared the rates of the enzyme catalysis by the above two methods. The experimental data revealed that the rate measured via the direct method was about twofold higher than that measured by the coupled-assay system. The above difference was found to be due to the inhibition of the enzyme by phenol, one of the reagents of the coupled assay system. The inhibitory role of phenol is not unique for indolepropionyl-CoA as substrate, but is also evident with aliphatic acyl-CoA substrates of varied chain lengths. Since the magnitude of inhibition is dependent on the nature of the acyl-CoA substrate, it is suggested that the coupled-reaction conditions must be carefully standardized with individual substrates. Some tips on standardizing the reaction conditions for quantitative measurement of the acyl-CoA oxidase-catalyzed reaction are offered.
Gopalan
K V
KV
Biochemistry Department, North Dakota State University, Fargo 58105, USA.
Srivastava
D K
DK
D000214:Acyl Coenzyme A / Q000032:analysis / Q000378:metabolism; D042962:Acyl-CoA Oxidase; D000675:Ampyrone / Q000494:pharmacology; D002175:Candida / Q000201:enzymology; D002733:Chlorophenols / Q000494:pharmacology*; D006735:Horseradish Peroxidase / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000032:analysis; D007700:Kinetics; D010088:Oxidoreductases / Q000037:antagonists & inhibitors*; D019800:Phenol; D010636:Phenols / Q000494:pharmacology*; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
D000214:Acyl Coenzyme A; D002733:Chlorophenols; D010636:Phenols; D000675:Ampyrone; C077841:3-indolepropionyl-coenzyme A; C077843:3-indoleacryloyl-coenzyme A; D019800:Phenol; D006861:Hydrogen Peroxide; D010088:Oxidoreductases; D006735:Horseradish Peroxidase; D042962:Acyl-CoA Oxidase; C004762:2,4-dichlorophenol