ENZYMES AS REAGENTS 



Another example of a microcyclic sequence of interactions is 

 catalyzed by giyceraldehyde-3-phosphate dehydrogenase and 

 its pyridine nucleotide coenzyme (Figure 3). 



DPN combines with the SH-enzyme to form a DPN-enzyme 

 complex (DPN-E) which can be measured by its absorption 

 band at 360 lUfx. The aldehyde interacts with the DPN-enzyme 

 to yield acyl enzyme and reduced DPN. In the presence of 

 phosphate, the acyl enzyme is phosphorolyzed to acyl phosphate 

 and SH-enzyme, which combines again with DPN to regenerate 

 the DPN-enzyme complex (38). 



ALDEHYDE DPN-E 



DP N H + H* 



l+ACCEPTOR) 



+ PHOSPHATE 



A C Y L- P 



Figure 3. 



The enzyme crystallizes from rabbit muscle in the presence 

 of ethylenediamine tetraacetate as the DPN-enzyme complex 

 with 3 moles of DPN per mole. After removal of DPN by 

 treatment with charcoal, the enzyme is somewhat less stable and 

 crystallizes only with difficulty. Exposure of the charcoal- 

 treated enzyme to acetyl phosphate results in the formation of 

 acyl enzyme. Crystallization of the protein from this mixture 

 occurs again quite readily and the crystalline material contains 

 the acetyl enzyme: It gives a positive hydroxylamine test and 

 oxidizes DPNH, thereby forming free acetaldehyde from the 

 acetyl group. With 1,3-diphosphoglycerate, the formation of a 

 phosphoglyceryl enzyme has been demonstrated (26). 



231 



