582 2. ANALOGS OF ENZYME REACTION COMPONENTS 



for aminopterin and the enzyme from Streptococcus faecalis, and Nath and 

 Greenberg (1962) gave K, as 0.0000023 mM for amethopterin and the calf 

 thymus enzyme. The inhibitions of the full reduction of folate to tetrahy- 

 drofolate by aminopterin and amethopterin are very similar (Futterman, 

 1957; Futterman and Silverman, 1957; Zakrzewski and Nichol, 1958; Silber 

 et al., 1962), and point to the dihydrofolate reductase as being the more 

 sensitive enzyme. Furthermore, the formation of folinate (citrovorum fac- 

 tor) from folate is very potently inhibited in rat liver slices (Nichol and 

 Welch, 1950), Lactobacillus casei and Streptococcus faecalis (Hendlin et al., 

 1953), mouse leukemic cells (Nichol, 1954), and chicken liver extracts 

 (Doctor, 1958). The most potent pteridine analog was found by Doctor 

 (1958) to be 2,4,7-triamino-6-o-methylphenylpteridine, although it is not 

 as potent as aminopterin, and he showed that the site of inhibition is pre- 

 vious to tetrahydrofolate. There is thus much evidence that folate reduc- 

 tion is blocked by low concentrations of these analogs, and it is generally 

 agreed that this must be the primary mechanism by which folate defi- 

 ciency and growth depression are produced. 



The inhibitions of folate reduction by aminopterin and amethopterin have 

 been reported to be noncompetitive by several investigators, but it is very 

 likely that the inhibitions are truly competitive, this being obscured by the 

 much greater affinity of the enzyme for the analog than for dihydrofolate. 

 In no case has the rate of inhibition in the presence of varying concentra- 

 tions of substrate been determined, but one might predict that the competi- 

 tive nature of the inhibition would be demonstrated in this way. Once the 

 enzyme is inhibited, it is very difficult to recover the activity because the 

 rate of dissociation of the analog from the enzyme is extremely slow. In 

 other words, this is an example of pseudoirreversible inhibition obeying mu- 

 tual depletion kinetics. This was shown by Peters and Greenberg (1959) on a 

 sheep liver folate reductase, the inhibition at constant analog concentration 

 being dependent on the enzyme concentration. It is thus possible to titrate 

 this enzyme in tissues or extracts. This has been well discussed by Werk- 

 heiser (1961), who also showed that the amount of analog bound by rat 

 liver supernates is equivalent to the amount required to inhibit folate re- 

 duction; in other words, the tightly bound analog seems to be combined 

 only with dihydrofolate reductase. If rats are injected with amethopterin, 

 the supernatant fraction of the liver contains most of the analog and only 

 10-15% of this is lost during dialysis for 6 days (Werkheiser, 1959). The 

 amount of amethopterin or aminopterin to inhibit completely the reductase 

 in liver extracts in 0.56 //g/g of liver, and the supernates of livers from 

 analog-treated rats contain 0.52 //g/g of tissue. The reductase is the only 

 protein binding these analogs significantly in chicken liver homogenates 

 (Schrecker and Huennekens, 1964). Fountain et al. (1953) had found that 

 there is a remarkable retention of amethopterin in the tissues of mice, the 



