MULTIPLE ENZYME SYSTEMS 



some of the problems which arise in approaching such reaction 

 systems it may be useful to consider the previously outlined MES 

 scheme under various conditions. 



Consider the situation: the reactions A -^ J have a lower 

 rate than J — * P. J will not accumulate under these circum- 

 stances. Normal procedure calls for an inhibitor or for frac- 

 tionation. Inhibitors can be very effective. Hartman, Leven- 

 berg, and Buchanan (15) recently employed azaserine, a known 

 broad-spectrum antibiotic, in the system synthesizing inosinic 

 acid and an aliphatic compound, formylglycinamide ribotide, ac- 

 cumulated. Inhibitors can be considered at various points in 

 the reaction. Thus metal-complexing agents, substrate-level in- 

 hibitors, cofactor inhibitors, and agents which might interrrupt a 

 regeneration mechanism should be considered. Merely treating 

 the extract by dialysis, ion-exchange resin, charcoal, heating, or 

 aging may remove cofactors or an undesired enzyme. 



Success of an enzyme fractionation certainly depends on the 

 complexity of the enzyme system, namely, the number of en- 

 zymes involved and the integration of the system with another 

 coupled reaction (s). Experience with glycolysis has clearly 

 shown that an interference with one of the carrier systems 

 (adenine or pyridine nucleotides) so that the acceptor or the 

 donor concentration decreases to levels which are too low for 

 optimal rates causes a precipitous fall in the overall rate of gly- 

 colysis. Such internally coupled systems generally are very sen- 

 sitive to changes, and a decrease in activity may begin a vicious 

 cycle which quickly results in a termination of the reaction. 

 Therefore best results might be expected in the fractionation 

 of perhaps a two-step reaction to which any needed coupled sys- 

 tem has been added. As usual such fractionations require ad- 

 equate analytical tools. We have found it useful to analyze 

 both the disappearance of substrate and appearance of the final 

 product, since this may give a measure of the isolation of the sys- 

 tem and the accumulation of an intermediate. 



A common approach to fractionation has been the separation 

 of the extract into two parts, say A and B. This procedure is 



549 



