NUCLEOTIDES AND COENZYMES 



Potter (22) has pointed out, it is precisely this competition by 

 alternate pathways for limited amounts of key substances and the 

 proper balance between energy production and utilization that 

 may well determine the precarious balance between normal and 

 uncontrolled growth. 



Recognition of a coenzyme as part of an enzymatic process 

 has been accomplished in a variety of ways. Often excellent 

 inferential evidence is gained from experiments in the field of 

 nutrition. Thus, Elvehjem (1) many years ago showed that 

 riboflavin deficiency in rats results in a decreased level of suc- 

 cinic dehydrogenase in the liver. This implication of a flavin 

 coenzyme in succinic dehydrogenase was verified only recently 

 upon isolation of the highly purified enzyme by Green et al. (5) 

 and by Singer and Kearney (38). The connection of a folic- 

 acid coenzyme with reactions involving one-carbon groups was 

 likewise obtained initially from vitamin-deficiency experiments. 

 Sometimes preliminary evidence has been elicited from experi- 

 ments where coenzyme or vitamin analogues are added to crude 

 systems with the resultant inhibition of a specific enzyme. 



By far the most convincing, and hence most often used, 

 criterion for coenzyme function is to obtain the enzyme free 

 from its coenzyme (i.e., the apoenzyme), and then to demon- 

 strate the absence of activity until the addition of the coenzyme.* 

 In a dissociated system the coenzyme becomes a second sub- 

 strate whose kinetic behavior can be analyzed by the conven- 

 tional Michaelis-Menten treatment or its extensions. The 

 Michaelis constants, or K^ values, so obtained are a general, but 

 not exact, measure of the aflinity of enzyme for coenzyme. 



With certain of the coenzymes, notably the flavin nucleo- 

 tides and pyridoxal phosphate, it is sometimes difficult to dis- 

 sociate the tightly bound coenzyme without concomitant de- 

 struction of the apoenzyme. In this event, it is usually pre- 

 sumptive evidence if the coenzyme can be shown to be present, 



* Resolved systems of this sort, usually with purified apoenzymes, pro- 

 vide an excellent assay for the particular coenzyme even in crude tissue ex- 

 tracts. 



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