SIDNEY F. VELICK 121 



well as in the redox step and that orientation may shift as a func- 

 tion of reaction conditions. Orthojjhosphate ions produce some en- 

 hancement (35) but do not alter the observed fluorescence polarization. 



S(}('ctr(il shifts. In view of the similarity in the fluorescence spectra 

 of DPNH in methyl carbitol and in the LDH complex, we can only 

 state that in the complex the coenzyme behaves as though it were 

 dissolved in a non-aqueous solvent of low or medium polarity. Specific 

 effects are undoubtedly involved, but the emission spectrum tells us 

 little about them. Absence of a shift in the GPD complex is again 

 consistent ^\ith its other properties including the apparent binding 

 in a closed conformation. 



Relative affinities of DPN and DPNH. The differences in free 

 energy of complex formation of enzyme with reduced and oxidized 

 forms of the coenzyme are determined by changes in conformation 

 and charge distribution that accompany oxidation and reduction. In 

 most cases the /\F° of complex formation is 2 to 3 kcal more negative 

 for DPNH than for DPN. The binding of DPN in several such cases 

 is strongly promoted by inhibitory substrate analogues, and presuma- 

 ably by substrate (5, 12, 17) , with the formation of an inhibitor-DPNH- 

 enzyme adduct. It was proposed previously that the inhibitor induces 

 a conformation in DPN that resembles that of DPNH, for which 

 the enzyme has a stronger affinity (35) . This type of interaction 

 should be at the core of the reaction mechanism, and the reader 

 is referred to a recent example produced by Winer and Theorell 

 (43) . In this property, too, GPD is exceptional in that in the absence 

 of substrate it binds DPN more strongly than DPNH and with a 

 A^° 3 to 5 kcal mole~^ more negative than is exhibited by other 

 enzymes. To account for this we can invoke conformation and group 

 interactions, but cannot specify the details. 



Enzyme specificity at pyridine-C-4. It was pointed out earlier that 

 the difference in the non-enzymatic reactivity of the two hydrogen 

 atoms at pyridine-C-4 of DPNH could be accounted for by the fact 

 that they have different chemical environments in the folded inner 

 complex. This influence should, and does, disappear when the di- 

 nucleotide is bound to an enzyme in an open conformation. In- 

 dividual enzymes of this group are seen to be specific for either 

 side of the pyridine ring, the specificity being imposed entirely by 

 the properties of the protein. The reactive hydrogen in the GPD 

 coenzyme complex, on the other hand, might be expected to be the 

 same as that of the free coenzyme in its folded conformation, but 

 this is not the case. The opposite stereospecificity is observed. Ob- 



