INHIBITIONS BY COENZYME ANALOGS 483 



volving ATP and a kinase. The analogs are occasionally phosphorylated 

 and exert their major effects in this form. The inability of most phosphor- 

 ylated substances to enter cells readily makes it necessary to use the analog 

 of the coenzyme precursor if inhibition in cell preparations is to be obtained. 

 Thus the initial analog or any of its metabolic products may interfere in a 

 number of reactions involving the coenzyme, and it is this that militates 

 against facile interpretations from superficially simple results. It should also 

 be evident that when the reaction catalyzed by the coenzyme-dependent 

 enzyme (A ^ B in Fig. 2-16) is determined, the kinetics of inhibition by 

 an analog of the coenzyme precursor will generally not be simple and, al- 

 though the fundamental block may be strictly competitive, the quantita- 

 tive relationship between the analog and the precursor will not necessarily 

 be competitive. 



The direct effect of a coenzyme analog on the enzyme reaction requiring 

 the cooperation of the coenzyme will depend on the tightness with which 

 the coenzyme is bound to the enzyme. Some coenzymes are so tightly bound 

 that they remain on the enzyme through numerous isolation procedures, 

 and in such cases the addition of an analog, even though it has a high 

 affinity for the enzyme, may not be able to replace the natural coenzyme 

 rapidly enough to induce inhibition. It must be remembered that the analog 

 does not actively displace the coenzyme (i.e., it does not force it from the 

 enzyme) but only binds to the free enzyme; if essentially all of the enzyme 

 is combined with coenzyme, there is little opportunity for the analog to act. 

 For this reason experiments on coenzyme analogs are frequently done with 

 reconstituted enzymes. In such cases the enzyme and coenzyme are disso- 

 ciated by some means and the effect of the analog on the reconstitution of 

 the active enzyme is investigated, this allowing the analog to act on the 

 free enzyme and to demonstrate competitive behavior. This technique is 

 not, of course, so applicable to cellular systems. 



It has been frequently stated that coenzyme analogs are specific inhi- 

 bitors. This is true in one sense inasmuch as these analogs or their meta- 

 bolic derivatives appear to interfere only with those enzymes or reactions 

 involving the corresponding normal coenzymes, in most instances. On the 

 other hand, the coenzymes often participate in several different types of 

 metabolic activity so that the metabolic disturbances produced by the 

 analogs may not be specific with respect to a single reaction. For example, 

 analogs of pyridoxal seem to interfere specifically with pyridoxal metab- 

 olism or the functions of pyridoxal phosphate, but pyridoxal phosphate 

 plays a role in many reactions of amino acids — racemization, transamina- 

 tion, oxidative deamination, decarboxylation, hydrolytic cleavage — as well 

 as being an important component of other enzyme systems, such as muscle 

 phosphor ylase, so that a deficiency of pyridoxal phosphate can induce wide- 

 spread disturbances. In addition to this, a generalized depression of amino 



