. Cell chemistry; a collection of papers dedicated to Otto Warburg on the occasion of his 70th birthday. Warburg, Otto Heinrich, 1883-; Biochemistry. VOL. 12 (1953) ENZYMES OF FATTY ACID METABOLISM 309 has been found***^ to be ^^ pjj ^ q -pj^g gj^. zyme does not react with free acetoacetate or with ethyl acetoacetate; it also fails to react with S-aceto- acetyl glutathione. With acetoacetyl-S-CoA the reac- tion is much faster than with the thioethanolamine derivative. This is undoubtedly due to the much higher affinity of the enzyme for the natural compound. In tact, in kinetic studies
. Cell chemistry; a collection of papers dedicated to Otto Warburg on the occasion of his 70th birthday. Warburg, Otto Heinrich, 1883-; Biochemistry. VOL. 12 (1953) ENZYMES OF FATTY ACID METABOLISM 309 has been found***^ to be ^^ pjj ^ q -pj^g gj^. zyme does not react with free acetoacetate or with ethyl acetoacetate; it also fails to react with S-aceto- acetyl glutathione. With acetoacetyl-S-CoA the reac- tion is much faster than with the thioethanolamine derivative. This is undoubtedly due to the much higher affinity of the enzyme for the natural compound. In tact, in kinetic studies with S-aceto- acetyl-N-acetyl thioethanolamine it was not pos- sible to reach saturation of the enzyme with the analogue^^. In the presence of thiolase, ^-keto reductase, and DPNH, the latter is oxidized on addition of acetyl-S-CoA; DPN+, j8-hydroxybutyryl-S-CoA and HS-CoA are the reaction products^. This occurs according to the reactions below: a. e CO -1-. ? 2 4 6 8 10 12 MINUTES Fig. g. Optical /?-keto reductase test. Pyrophosphate buffer pH , 50 ixM; DPNH, fxH; S-aceto- acetyl-N-acetyl thioethanolamine, jxM. Volume, ml; d = cm; temp. 25°. O 2CH3—C—S—CoA: O HS—CoA+CH3—CO—CH 2—O—S—Co A (thiolase) O CH3—CO—CHj—C—S—C0A + DPNH + H+ ^ CH3—CHOH—CH,—C—S—C0A + DPN+ (reductase) O Sum: 2 CH3—C—S—Co A + DPNH + Hh O II CH3—CHOH—CH2—C—S—CoA + HS—CoA + DPN+ The formation of HS-CoA can be followed through the appearance of sulfhydryl groups. /?-hydroxybutyryl-S-CoA was extracted from the acidified reaction mixture with /j-cresol and converted into the corresponding hydroxamic acid by reaction with hydroxylamine. The j3-hydroxybutyrohydrox- amic acid was identified by paper chro- matography {Rp in aqueous butanol, ). On incubation of the natural j8-hydroxy- butyryl-S-CoA with DPN+ and purified ^- ketoreductase at pH , the reduction of DPN+, followed at 340 m/x, is accompanied by the formation of acetoacety
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