ig6 A. L. LEHNINGER, G. D. GREVILLE VOL. 12 (1953) 



Rp under the same conditions of 0.35. No other hydroxamic acid spots were detected 

 on such papergrams of the reaction products. From the now very extensive evidence 

 that this ATP- and CoA-dependent trapping reaction indicates the formation of acyl- 

 CoA conjugates, it may be concluded that the j8-hydroxybutyryl-CoA complex is in 

 fact formed in these extracts. 



Although the chromatographic method identified the hydroxamic derivative as 

 that of ^-hydroxybutyric acid it was of course uncertain whether this was present as the 

 CoA complex of the d- or the /-isomer. Additional evidence was collected however which 

 leaves very little doubt that the ^-isomer is present in this complex although the direct 

 measurement of the rotation of pure /S-hydroxybutyryl CoA has not been made. The 

 first point of evidence bearing on this problem is that /-BOH, which docs not undergo 

 significant oxidation by DPN+ in the extracts used in experiments of Table VI does 

 nevertheless also form a hydroxamic acid derivative under the same circumstances as 

 d-BOH (see Table VII). This formation is also absolutely dependent on ATP and CoA but 



TABLE VII 



HYDROXAMIC ACID FORMATION FROM d- AND /-BOH 



System contained 0.3 ml extract (3-C-7), o.i M KCl, o.oi M cysteine, 0.05 M "tris" buffer 

 pH 8.0, 0.5 71/ hydroxylamine (brought to pH 8.0), 0.025 M d- or Z-BOH, 0.005 ^I ATP. 0.005 M 

 MgCU, and o.ooi M CoA. Total volume, i.o ml. Time, 60 minutes; temp. 22° C. 



Substrate Svstem Hydroxamic acid 



•^ formed micromoles 



has been found to proceed at a slightly lower rate than that from the ^-isomer (the dif- 

 ference in rate may not be significant). Furthermore, by paper chromatography it was 

 established that this hydroxamic acid moves at the same rate as that formed from 

 i/-BOH on Whatman No. 43 paper using the butanol-water system. It would appear 

 therefore that reaction (3) is not very specific stereochemically and that both isomers 

 form CoA derivatives in the presence of enzyme, ATP and CoA. However it must be 

 pointed out again that only the (/-isomer causes reduction of DPN+ in the oxidation 

 test system. It would appear then that although the /-j8-hydroxybutyryl-CoA complex 

 is also formed, it does not cause reduction of DPN+ in these extracts. The dehydrogenase 

 catalyzing reaction (4) is tlierefore probably specific for the f/-/S-hydroxybutyryl-CoA. 

 Such experiments also indicate that the /-^-hydroxybutyryl-CoA does not undergo 

 enzymic racemization in these extracts at any significant rate. 



Other points of evidence may be summarized. Both crotonate and vinyl acetate, 

 when substituted for d-BOW in the complete test system, cause reduction of DPN+ 

 at about the same rate as does (/-BOH. In each case there is an absolute requirement 



References p. 202. 



