NUCLEOTIDES AND COENZYMES 



pyridine nucleotides are firmly bound in vivo to their apoenzymes, 

 or whether they form only transient complexes enabling them to 

 shuttle between two dehydrogenases of a linked reaction, as in 

 glycolysis, for example. The fact that pyridinoproteins, ex- 

 cept 3-phosphoglyceraldehyde dehydrogenase, have been iso- 

 lated as completely dissociated enzymes, with the corollary 

 finding that most Km values for the reconstituted enzymes are 

 relatively high, argues in favor of the latter hypothesis. How- 

 ever, Huennekens and Green (20) have presented evidence that 

 many of the pyridinoproteins within mitochondria appear to be 

 conjugated with respect to DPN, and upon disorganization of 

 the mitochondrial architecture, destruction or dispersion of the 

 DPN causes a requirement for added DPN to appear. In 

 addition it has been shown recently that the tightly bound DPN 

 of 3-phosphoglyceraldehyde dehydrogenase can be utilized, 

 apparently without being dissociated, in a linked reaction with 

 lactic apodehydrogenase, or as a substrate for DPN-cytochrome 

 c reductase. 



In addition, it should be remarked that there is a consider- 

 able body of evidence scattered throughout the literature to the 

 effect that in certain cases a coenzyme may be bound originally 

 in a one-to-one ratio with its protein, but after dissociation has 

 once occurred, rebinding of the components may be of a de- 

 generate type where a great excess of coenzyme must be present. 



In the field of flavoproteins early work by Kuhn implicated 

 the N-3 position in the isoalloxazine ring and the phosphate 

 group as binding sites. More recently, Mahler (31) has sug- 

 gested a more detailed picture to explain the respective bindings 

 in metalloflavoproteins. Even the seemingly innocuous sugar 

 moieties may be more than insulating groups and may participate 

 in binding. Huennekens and Snell (21) have found that the 

 lyxoflavin analogues of FMN and FAD have noticeably higher 

 K^ values than the parent compounds when compared in 

 enzymatic assay systems. Recent findings have revealed the 

 surprising fact that FAD, bound to succinic dehydrogenase, is 

 not entirely released by trichloracetic acid or heat denaturation 



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