3o8 



F. LYNEN, S. OCHOA 



VOL. 12 (1953) 



non-labelled acetoacetyl-S-CoA from the last four carbons of the fatty acid chain reacts 



with thiolase to give non-labelled acetyl-S-enzyme and this in turn reacts with labelled 



O 



acetyl-S-CoA from the pool to yield CH3— CO— CH,— C* — S— CoA. This is shown schemat- 

 ically in Fig. 8 for the case of caproic acid. 



O 



CH, — CH,— CH., — CO- 



O 



CHj — 'CHo — CHg — 'C- 



O 



-CHj— C — S— CoA 



(HS— Enz) O 



-S— Enz CH3— C — S— CoA 



(HS— CoA) 



CH3— CH, — CH.,— C— S— CoA HS— Enz 



CH3— C 



CH3— CO— CHj— C *— S— Co A + HS— Enz 



l(H,0) 

 CH3— CO— CH,— C*OOH + HS— CoA 



CH3— C*— S — Enz + HS — CoA 

 O 



(CH3 — C*— S — CoA 

 O 



CH3— C*0— CH„— C*— S— CoA 



J 



(H^O) 

 CH3— C*0— CH,— C*OOH + HS— CoA 

 Fig. 8. Asymmetric labelling of acetoacetate from carboxyl-labelled caproic acid. 



^-keto-redudasc. This enzyme catalyzes Reaction 6. The finding that 



\ 



o 



CH3— CO— CH2— C— S— C0A + DPNH + H+ ^ CH3— CHOH— CHg- C— S— C0A+DPN+ (6) 



the acetoacetyl-S-CoA analogue, S-acetoacetyl-N-acetyl thioethanolamine, was readily 

 reduced by DPNH in the presence of enzyme solutions from various sources afforded a 

 convenient assay for this enzyme. Employing this assay the enzyme was purified some 

 300-fold from sheep liver extracts by a procedure involving three steps : precipitation 

 with ethanol, denaturation of inactive protein at 55°, and fractionation with am- 

 monium sulf ate^^. The time course of the reaction in the optical test with varying concen- 

 trations of the purified enzyme is shown in Fig. 9. 



The reaction is readily reversible but, at pH 7.35 with equimolccular amounts of 

 DPNH and the acetoacetyl thioethanolamine derivative, it proceeds in the direction 

 of reduction of the latter to the extent of 95 %. The equilibrium constant of the reaction 

 (/iTe'q = (S-i3-hydroxybutyryl compound) (DPN+)/(S-acetoacetyl compound) (DPNH)) 

 References p. 313J314. 



