VOL. 12 (1953) ENZYMIC SYNTHESIS AND BREAKDOWN OF OROTIC ACID I 



229 



0.600 



0.500 



O.WO 



o 



7lfg+2. While the presence of added Mg+2 had Httle effect on the initial rate of the 

 reaction, an effect was apparent when the reaction proceeded for longer periods. Thus, 

 at o, 2-10-^ M, and 5-10-^ M Mg+2, the decrease in optical density at 280 m/i in 6 

 minutes was found to be 0.225, 0.240, and 0.261, respectively, whereas at 20 minutes 

 the density decreases were 0.355 ,0426, and 0.503. 



Cysteine. Freshly prepared cell- 

 free extracts showed httle or no stim- 

 ulation upon the addition of cys- 

 teine. The reaction rate with the 

 purified enzyme preparation, how- 

 ever, was increased up to twofold in 

 the presence of 0.002-0.007 M cys- 

 teine. Larger amounts of cysteine 

 appeared to have an inhibitory effect. 



Oxidation of dihydro-orotate to § 0.300 

 orotate. The reversibility of the con- >. 

 version of orotate to dihydro-orotate 

 was demonstrated with the purified 

 enzyme. Dihydro-orotate (0.263 mi- 

 cromole) was incubated with. 26 units 

 of the enzyme under the usual assay 

 conditions except that glucose and 

 glucose dehydrogenase were omitted. 

 The oxidation of reduced DPN was 

 presumably achieved by the action 

 of DPNH oxidase which was present 

 in the enzyme preparation. A sub- 

 stance having the absorption spectrum 

 of orotate was formed from dihydro- 

 orotate in theoretical yield and com- 

 plete removal of this material could 

 be effected by the addition of glucose 

 and glucose dehydrogenase (Fig. 2, 3). 



0.200 



^ 0.100 



0.00 



100 



Time in minutes 



Fig. 2. Theoxidation of dihydro-orotate toorotate. 

 The incubation mixture in the experimental 

 cuvette contained 15 micromoles of MgClg, 100 

 mici^omoles of potassium phosphate buffer (pH 

 6.1), 30 micromoles of cysteine (pH 7.0), 26 units 

 of dihydro-orotic dehydrogenase (specific activity 

 184), 0.05 micromole of DPN, and 0.263 micro- 

 mole of dihydro-orotic acid (enzymic origin) in 

 a volume of 3.0 ml. The incubation mixture in 

 the blank cell contained no dihydro-orotic acid. 

 250 units of glucose dehydrogenase and 200 

 micromoles of glucose were added at the times 

 indicated. 



Unlike the reduction of orotate 

 to dihydro-orotate, which does not proceed in the absence of added DPN, the oxid- 

 ation of dihydro-orotate by the same purified enzyme preparation was observed to 

 occur at the same rate with or without added DPN. 



In order to establish that dihydro-orotate oxidation does involve DPN reduction 

 the reaction was studied under anaerobic conditions and in the presence of pyruvate 

 and lactic dehydrogenase. As shown in Fig. 4, little orotate synthesis was observed in 

 the absence of a DPNH oxidant such as the oxygen-DPNH oxidase or the pyruvate- 

 lactic dehj/drogenase systems. 



The non-identity of natural dihydro-orotic acid and the synthetic fusion product. The 

 fusion product of maleic acid and urea was considered to be dihydro-orotic acid by 

 Bachstez and Cavallini^o, although they offered no proof of structure. This compound 

 can be compared to the product of enzymic reduction of orotic acid in the following 

 respects: 

 References p. 234. 



