ANALOGS OF THIAMINE 519 



some exchange between bound and free coenzyme and analog must occur, 

 it is too slow for equilibrium to be obtained easily. To determine the maximal 

 inhibiting power of an analog it is advisable to incubate the apoenzyme with 

 the analog previous to addition of the coenzjTne. 



Pyruvate oxidase is inhibited similarly to the decarboxylase, as expected, 

 and in the case of pyrithiamine-PP it would appear to be competitive 

 (Woolley, 1951). Oxythiamine-PPP inhibits pyruvate oxidation in pigeon 

 breast muscle extracts but this may be mediated through the diphosphate 

 (Onrust et al., 1952). The formation of acetoin from pyruvate is also inhib- 

 ited by oxythiamine-PP (Eich and Cerecedo, 1954). However, Kuratomi 

 (1959) noted that oxythiamine, like thiamine, can form acetoin from py- 

 ruvate. 



Transketolase from yeast is strongly inhibited by oxythiamine-PP (Datta 

 and Eacker, 1961) but oxythiamine itself has no effect (Dreyfus and Moniz, 

 1962). At 0.036 mM and 0.072 mM the inhibitions are 60% and 80%, re- 

 spectively, when the analog is added previous to thiamine-PP, but if the 

 oxythiamine-PP is added 2 min after the coenzyme, no inhibition is ob- 

 served. Addition of higher concentrations of thiamine-PP cannot reverse 

 the inhibition. Thus it is difficult for either the analog or the coenzyme to 

 displace each other from the apoenzyme. Oxythiamine-PP is bound more 

 tightly than thiamine-PP to the enzyme but it requires 2-3 hr to inhibit 

 50% when the enzyme initially contains thiamine-PP. The rate of dis- 

 placement for transketolase is even less than for decarboxylase, since 

 thiamine-PP was found to reverse oxythiamine-PP-inhibited enzyme 30% 

 in 20 min. 



The inhibition of thiamine-PP-dependent enzymes by oxythiamine and 

 pyrithiamine will depend on whether these analogs can be phosphorylated 

 or not. Thus in the experiments of Kunz (1956), where oxythiamine and 

 pyrithiamine at 10 vaM inhibited pyruvate oxidation in rat liver mitochon- 

 dria 95% and 35%, respectively, one is not certain if there is direct inhibi- 

 tion or if the depression was due to the formation of small amounts of the 

 phosphorylated esters. Acetylthiamine inhibits to about the same degree 

 as pyrithiamine and this analog cannot be phosphorylated (unless it is first 

 deacetylated), so it would seem that these rather weak inhibitions may be 

 to some extent exerted directly. It is also interesting that oxythiamine 

 and pyrithiamine have no effect on pyruvate oxidation in brain mitochon- 

 dria, this being attributed by Kunz to a different structure or permeability 

 compared to liver mitochondria; different phosphorylative capacities might 

 also play a role. Phosphorylation of these analogs mediated through thia- 

 mine kinase seems to occur in most tissues and it is likely, as pointed out 

 by Woolley (1951), that the toxic reactions observed with oxythiamine 

 and pyrithiamine in animals are produced primarily by the phosphorylated 

 compounds. 



