BENT LEY GLASS 839 



the others, Hiiorescence is partially queiuhed. The cxtilaiion spec- 

 trum is strongest at 280 niyu,, where it is attributable almost wholly to 

 the protein rather than the coenzyme. The emission, at 490 m/x, 

 comes from the coenzyme; and accordingly, there must be a transfer 

 of excitation energy from the protein to the bound coenzyme. With 

 GPD, and with GPD alone, there is partial depolarization of the 

 fluorescent light, as if the bound coenzyme possessed some degree 

 of oscillatory freedom. Another distinction is that in the case of 

 GPD the product of substrate oxidation remains bound to the enzyme 

 until it undergoes a second reaction step, a phosphorylation, which 

 is also influenced by the bound coenzyme. Maybe the DPNH is only 

 rigorously fixed in position in the presence of the appropriate 

 substrate. 



Enzymes which shift the maximum absorption of bound DPNH 

 toward the shorter wavelength end of the spectrum also shift the 

 fluorescence emission band of the DPNH in the same direction; but 

 no simple proportionality is to be observed, and the enzymes of this 

 class (like LDH and unlike GPD) are not to be thought of as form- 

 ing identical sorts of complexes. The relatively low affinity of the 

 LDH class of enzymes for DPN may be greatly enhanced by nucleo- 

 philic reagents (such as cyanide, bisulfite, or hydroxylamine) which 

 attack the pyridine C-4 atom and possibly convert it to a form re- 

 sembling DPNH. These enzymes like LDH do not generate a dif- 

 ferential reactivity of the two H atoms on C-4 of the pyridine ring, 

 since the coenzyme is bound to them in open conformation. In the 

 case of GPD, where the stereo-specificity is present, the reactive H 

 atom is most luiexpectedly not the one identified as such in the free 

 but folded DPNH. The change to an opposite stereospecificity, ob- 

 served here, should have something to do with the structure of the 

 inner complex. Perhaps, as Velick suggests, a face to face or edge to 

 edge alignment of the adenine and pyridine rings, an arrangement 

 unstable in water even with the help of the postulated hydrogen 

 bond (adenine amino to nicotinamide carbonyl oxygen) , might be 

 stabilized in the enzyme complex. 



Of the analogues, desamino-DPNH is actually a better coenzyme 

 with LDH than is DPNH, although desamino-DPNH has low activity 

 in the GPD reaction. It is bound mainly in the open conformation. 

 On the other hand, the acetyl pyridine analogue, which is perhaps 

 bound in the folded conformation (for it occurs in that form in 



