424 



2. ANALOGS OF ENZYME REACTION COMPONENTS 



to establish their role in metabolism that Karunairatnam and Levvy (1949) 

 searched for specific inhibitors. The most potent of the analogs tested is 

 glucarate (K^ = 0.06 mM, and K,^, = 3.5 for phenyl-/?-glucuronide) but 

 subsequent studies in different laboratories showed great variability in ac- 

 tivity. The problem was solved by Levvy (1952), who found that the ac- 

 tive inhibitor is glucaro-l,4-lactone,* which is formed from glucarate and 

 occurs to varying extents in different preparations. The tabulation shows 



the inhibitor constants obtained on mouse liver /^-glucuronidase hydrolyz- 

 ing phenolphthalein-/?-glucuronide, and the particular potency of the glu- 

 caro- 1,4-lactone is evident."^ No inhibition at 1 mM is observed with man- 

 narate, mannaro-l,4-3,6-dilactone, 4-methylglucuronate, and ascorbate, or 

 at 10 mM with 3-methylglucarate, glucurone, 3-methylglucuronate, man- 

 nuronate, mannurone, and 2-keto-L-gulonate. Heat and acid treatments of 

 glucarate and galactarate increase the inhibitory activity greatly and it is 

 possible that the dicarboxylates are completely inactive. The inhibition by 

 glucaro- 1,4-lactone is competitive and the high potency probably due to 

 the structural similarity between the inhibitor and the /5-glucofuranuronide 

 form of the substrate. The hydroxyl configuration in the furan ring is very 

 important since the glucaro-3,6-lactone is bound much less tightly to the 

 enzyme, and the 3-OH must also be involved since methylation reduces 

 the binding by over 3 kcal/mole. These rather large energy differences argue 



* This substance has also been called saccharo- 1,4-lactone, 1 ,4-saccharolactone, 

 glucosaccharo- 1,4-lactone, and other names. The generic name for the dicarboxylic 

 acids derived from sugars is saccharic acid and that specifically from glucose is glu- 

 cosaccharic acid. However, it seems that glucaric acid is used most commonly today 

 and this terminology is more consistent with the naming of the monocarboxylic acids, 

 so that the lactones will here be named accordingly. 



t The figures in this and other reports for inhibitors such as glucarate, galactarate, 

 glucuronate, and related acids may reflect to varying degrees the presence of lactones, 

 since, due to the high potency of the lactones, only small amounts need be present. 



