228 



I. LIEBERMAN, A. KORNBERG 



VOL. 12 (1953) 



0.300 



Specificity of DPNH and influence of DPNH concentration on orotate reduction. 

 With the purified enzyme preparation no observable reduction of orotate occurred in 



the absence of added DPN. With TPN, 



M X 10^ orotate CS) , . , , . , 



5 10 15 20 25 30 the Tcaction rate was Icss than 2 % of that 



observed with DPN, and no inhibiting 

 effect on the reaction (with DPN) was 

 noted. 



The rate of orotate reduction ap- 

 peared to be dependent on DPNH concen- 

 tration since it was found to be propor- 

 tional to the amount of glucose dehydro- 

 genase when this enzyme was added in 

 limiting quantities. In one experiment, 

 with 9.5 units of purified enzyme, the 

 decrease in optical density at 280 mju, in 

 4 minutes was found to be 0.151, 0.301, 

 0.572, and 0.565, with 62.5, 125, 250, 

 and 310 units of glucose dehydrogenase, 

 respectively. With 4 units or less of di- 

 hydro-orotic dehydrogenase, glucose de- 

 hydrogenase at a level of 250 units did 

 not limit the rate of the reaction. The 

 concentration of glucose used in the assay 

 system was 0.067 ^^' ^^^ it appeared to 

 be sufficient to allow a maximum rate 

 of orotate reduction. Decreasing the con- 

 centration of glucose to 0.033 M caused 

 a decrease of less than 10% in the reaction 

 rate. Approximately half the maximum 

 rate was obtained with 0.008 M glucose. 

 Specificity of dihydro-orotic dehydro- 

 genase and the influence of orotate concen- 

 tration on rate of reaction. The possibility 

 that dihydro-orotic dehydrogenase can 

 catalyze the reduction of other pyrimidines was investigated. No activity and no 

 inhibition of orotate reduction was observed with uracil, cytosine, 5-methylcytosine, or 

 thymine. 



The rate of orotate reduction was studied as a function of orotate concentration 

 (Fig. i). When the data were plotted according to Lineweaver and Burk-", as shown 

 in the figure, a straight line was obtained. K^ was calculated to be i.i • lO"* M. 



Fig. I. The rate of orotate reduction as a 

 function of orotate concentration. 



The reaction mixtures containing 2.7 

 units of dihydro-orotic dehydrogenase (spe- 

 cific activity 86) were prepared as for the 

 standard assay (see Methods) except that 

 varying amounts of sodium orotate (molar 

 concentrations = S) were used. The progress 

 of the reaction was followed by measuring the 

 decrease in optical density at 280 m/t during 

 a 6 minute interval (v). The solid circles rep- 

 resent the decrease in optical density as 

 a function of orotate concentration. The open 

 circles represent a Lineweaver-Burk plot^'. 



The effect of pH and other factors on orotate reduction 



pH. The pH of the assay medium was 6.4-6.5. At pH 7.0, the initial rate of the 

 reaction was the same as at 6.5, but the enzyme appeared to be less stable and the rate 

 declined more rapidly than at the lower pH. At pH 5.5 and 7.8, the initial rates of reac- 

 tion were 13 and 65%, respectively, of that at pH 6.5. 

 References p. 234. 



