ENZYMES ATTACKING NUCLEIC ACIDS 621 



The enzyme reaction requires either the presence of stoichiometric amounts 

 of reduced DPN or of catalytic amounts of DPX in the presence of a hy- 

 drogen-donor system. 



Specificity. Dihydroorotic acid dehydrogenase is strictly specific for orotic 

 acid. Uracil, cytosine, thymine, and 5-methylcytosine are inert and have no 

 influence on the rate of hydrogenation of orotic acid. 



pH Optimum. The optimal pH range is between pH 6.4 and 6.5. 



The Michaelis-Menten constant has the value of 1.1 X 10~^ M concentra- 

 tion of orotic acid. 



The presence of the enzyme in other microorganisms is suggested by the 

 fact that dihydroorotic acid is a growth factor for Lactobacillus bulgaricus. 



b. Dihydroorotase^'^'^'^ 



The reversible hydrolysis of dihydroorotic acid to ureidosuccinic acid is 

 catalyzed by dihydroorotase Avhich is present in cell-free extracts of Zymo- 

 bacterium oroticum. 



Equilibrium. Lieberman and Kornberg measured the equilibrium of the 

 reaction by using C"-labelled substrates and by determining the total radio- 

 activity of the components of the incubation mixture after chromatographic 

 separation. At pH 6.1, the ratio of ureidosuccinate to dihydroorotate at 

 equilibriam is approximately 1.9. 



c. 5-Carboxymcthylhydantoinase-''^'' 



It is interesting that the formation of dihydroorotic acid is not the only 

 type of enzymic ring-closure which ureidosuccinic acid undergoes in cell- 

 free extracts of Zymobacterium oroticum. These extracts contain a specific 

 enzyme, 5-carboxymethylhydantoinase, which catalyzes the reversible ring- 

 closure between the ureido group and the vicinal carboxy group to l-5- 

 carboxymethylhydantoin. 



Equilibrium. The eciuilibrium constant is similar to that of dihydrooro- 

 tase, amounting to the value of 1.9 for the proportion between 5-carboxy- 

 methylhydantoin and ureidosuccinate. 



The specificity of 5-carboxymethylhydantoinase has not yet been investi- 

 gated. Liver homogenates which hydrolyze hydantoin-^^f were found to be 

 without activity toward L-5-carboxymethylhydantoin. 



2. Conversion of Dihydrouracil and Dihydrothymine to j8-Amino 

 Acids by Tissue Slices 



The reductive pathway of the reversible degradation of orotic acid under 

 the influence of the enzyme system of Zymobacterium oroticum has an 

 interesting parallel in the behavior of liver and kidney slices toward di- 

 hydrothymine and dihydrouracil. Fink et a/."^^ demonstrated by paper 



""F. Bernheim and M. Bernheim, J. Biol. Chem. 163, 683 (1946). 



2'^8 R. M. Fink, K. Fink, and R. B. Henderson, J. Biol. Chem. 201, 349 (1953). 



