IV. BIOCHEMICAL SYSTEMS 491 



In the oxidized state, the nicotinamide nucleus exists as a quaternary 

 pyridinium ion which forms an inner salt mth one of the ionizable acid 

 groups in the pyrophosphate bridge. Upon reduction the pyridine nitrogen 

 is converted to a weakly basic tertiary amine, hence changing the proper- 

 ties of the compound. 



The hydrogen-transferring property of DPN had been postulated in 

 1934^^ on the basis of its function as coenzyme for a number of dehy- 

 drogenases. It remained, however, for Warburg and associates in their 

 classic work^ • - ■ * to clearly demonstrate this mechanism in their studies 

 with TPN and later with DPN. 



This reaction probably proceeds in more than one step with the inter- 

 mediate formation of semiquinoid radicals. As shown by Warburg and 

 associates,^ when DPN or TPN are reduced by hydrosulfite, an intensely 



H H 



C C 



/ \ / \ 



HC C— CONH2 HHC C— CONH2 



1 II \l II 



HC CH + Na2S204 + 2H2O -> C CH -f 2NaHS03 + HX 



\ / / \ / 



N H N 



l\ I 



R X R 



yellow semiquinoid substance (monohydrocoenzyme) is observed as an 

 intermediate in the reaction. This intermediate substance can be stabilized 

 by carrying out the reaction in a strongly alkaline medium. ®^'®^ 



2. The Apoenzymes 



DPN and TPN function in biochemical reactions only when joined with? 

 or activated by, specific proteins (apocodehydrogenases) . Although the 

 exact mechanism by which the protein apoenzyme unites with the coen- 

 zyme is very unsettled, it is clear that the coenzyme will not function 

 catalytically without the influence of the apoenzyme. It has been postu- 

 lated that the apoenzyme functions by facilitating formation of the semi- 

 quinoid intermediates referred to above. ^^■^° 



Although the coenzymes are quite non-specific in that they function in 



6* H. von Euler, Chemie der Enzyme, Vol. II, Part 3. Bergman, Munich, 1934. 



" P. Karrer and F. Benz, Helv. Chim. Ada 19, 1028 (1936). 



" E. von Euler, H. Hellstrom, and H. von Euler, Hoppe-Seyler's Z. physiol. Chem. 



242, 225 (1936). 

 «8 F. Schlenk and T. Schlenk, Arch. Biochem. 14, 131 (1947). 

 69 L. Michaelis and C. V. Smythe, Ann. Rev. Biochem. 7, 1 (1938). 

 '" L. Michaelis, Advances in Enzymol. 9, 1 (1949). 



