A substitutive substrate for measurements of beta-ketoacyl reductases in two fatty acid synthase systems.
Bacterial beta-ketoacyl-ACP reductase (FabG) and the beta-ketoacyl reductase domain in mammalian fatty acid synthase (FAS) have the same function and both are rendered as the novel targets for drugs. Herein we developed a convenient method, using an available compound ethyl acetoacetate (EAA) as the substitutive substrate, to measure their activities by monitoring decrease of NADPH absorbance at 340 nm. In addition to the result, ethyl 3-hydroxybutyrate (EHB) was detected by HPLC analysis in the reaction system, indicating that EAA worked effectively as the substrate of FabG and FAS since its beta-keto group was reduced. Then, the detailed kinetic characteristics, such as optimal ionic strength, pH value and temperature, and kinetic parameters, for FabG and FAS with this substitutive substrate were determined. The Km and kcat values of FabG obtained for EAA were 127 mM and 0.30 s(-1), while those of this enzyme for NADPH were 10.0 microM and 0.59 s(-1), respectively. The corresponding Km and kcat values of FAS were 126 mM and 4.63 s(-1) for EAA; 8.7 microM and 4.09 s(-1) for NADPH. Additionally, the inhibitory kinetics of FabG and FAS, by a known inhibitor EGCG, was also studied.
Sun
Ying-Hui
YH
Department of Biology, Graduate University of Chinese Academy of Sciences, Beijing 100049, PR China.
Cheng
Qing
Q
Tian
Wei-Xi
WX
Wu
Xiao-Dong
XD
D064431:3-Oxoacyl-(Acyl-Carrier-Protein) Reductase; D000090:Acetoacetates / Q000378:metabolism; D000429:Alcohol Oxidoreductases / Q000032:analysis* / Q000378:metabolism*; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology; D064429:Fatty Acid Synthases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D015394:Molecular Structure; D009249:NADP / Q000378:metabolism; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D013379:Substrate Specificity; D013696:Temperature
D000090:Acetoacetates; D009249:NADP; D000429:Alcohol Oxidoreductases; D064431:3-Oxoacyl-(Acyl-Carrier-Protein) Reductase; D064429:Fatty Acid Synthases; C024840:ethyl acetoacetate