310 



F. LYNEN, S. OCHOA 



VOL. 12 (1953) 



of r^-/9-hydroxybutyryl-S-CoA by DPN+. The latter reaction, which is undoubtedly 

 catalyzed by the ^-ketoreductase here described, was demonstrated by making use of 

 the fact that liver also contains enzymes catalyzing the formation of d- or /-jS-hydroxy- 

 butyryl-S-CoA in the presence of ATP, CoA-SH, and d- or /-j8-hydroxybutyrate. 



The chain-length specificity of the ^-ketoreductase is still unknown and it is not 

 possible to decide at this time whether more than one enzyme is concerned with the CoA 

 derivatives of j3-keto and /^-hj^droxy acids from C4 to C^g. The purified reductase described 

 above has been found to act rapidly on S-/S-ketocaproyl-N-acetyl thioethanolamine^^^. 



Crotonaso. Synthetic S-crotonyl CoA is converted to S-acetoacetyl CoA, in the pres- 

 ence of DPN, by crude enzyme preparations from heart or liver^'''. The reaction can be 

 followed through the appearance of the absorption band of DPNH at 340 mju, or that of 

 acetoacetyl-S-CoA at 305 m/x. Also, on addition of HS-CoA, citrate condensing enzyme, 

 and oxalacetate, crotonyl-S-CoA acts as an acetyl donor for citrate synthesis; the 

 required thiolase was present in the crude enzyme preparation used. These observations, 

 together with the fact that reduced leucosafranine is oxidized by synthetic j3-hydroxy- 

 butyryl-S-CoA in the presence of partially purified preparations of ethylene reductase^^ 

 indicate the occurrence of an enzyme catalyzing the reversible Reaction 7 below. The 

 name crotonase has been suggeste.d for this enzyme^^. The enzyme has no action on free 



o o 



CH.,— CH = CH— C— S— CoA + HoO ^ CH.,— CHOH— CH„— C— S— CoA 



(7) 



0.4 



0.3 



0.1 



crotonate or on the S-crotonyl derivatives of N-acetyl thioethanolamine, glutathione or 

 thioglycolic acid. 



As already mentioned the spectrum of S-crotonyl CoA is similar to that of S-crotonyl- 

 N-acetyl thioethanolamine. This is readily apparent when the contribution of the adenine 

 moiety of the CoA derivative is eliminated by reading S- 

 crotonyl CoA against a solution containing an identical 

 amount of the compound but previously subjected to 

 alkaline hydrolysis. The difference spectrum so obtained^-^, 

 illustrated in Fig. 11, shows absorption maxima at 224 

 and 263 m/x like S-crotonyl-N-acetyl thioethanolamine, 'f 

 The crotonyl CoA was obtained through reaction of ^ 0.2 

 CoA-SH with crotonic anhydride following the method"J* 

 of Simon and Shemin^^. 



The decrease in light absorption at 263 mju when 

 crotonase acts on crotonyl CoA affords a simple method 

 of assay for this enzyme. The purification of the enzyme 

 from ox liver has recently been undertaken. Through 

 steps involving denaturation of inactive proteins by acidi- 

 fication and heat, followed by acetone, ammonium sulfate 

 and low temperature ethanol fractionation, preparations 

 of the enzyme have been obtained representing about 

 lOO-fold purification over the original extracf"^^. The 

 preparations are free of fumarasc showing that fumarase 

 and crotonase are distinct enzymes. Crotonase has a 

 remarkably high activity as may be seen in Fig. 12. which shows the time course of 

 the reaction in the optical test with varying amounts of the enzyme. The equilibrium 

 References p. 31 3 1 314. 



220 



2'.0 260 280 300 



WAVELENGTH (m^) 



Fig 



1 1 . Difference ultraviolet 

 absorption spectrum of S- 

 crotonyl CoA before and after 

 alkaline hydrolysis of the thio- 



ester bond, c 



6- 10-5 M in 



each cell; d = 0.5 cm; pH, 7.5. 

 Crotonyl CoA in blank cell pre 

 viously hydrolyzed with alkali. 



