98 



Kinetic constants 



Affinity purified SP-I and SP-II were used to 

 characterize the properties of the two proteins. Both 

 proteins were specific for their substrates and neither 

 enzyme would utilize NAD* as cofactor or quinate as 

 substrate. Saturation of the SDH activity domain of the 

 bifunctional enzyme with substrates, NADP* (at 0.14 mAf) and 

 SHK (at 5.0 mAf), is shown in Fig. 5-10 (A and C) for SP-I 

 and in Fig. 5-11 (A and C) for SP-II (saturating at 0.2 mM 

 NADP* and 4 . mAT SHK. From the double-reciprocal plots in 

 Figs. 5-10 and 5-11 (panels B and D) , the Km values for the 

 SDH domain of both SP-1 and SP-II were found to be 0.02 mM 

 for NADP*, However, different values for shikimate of 0.8 mAf 

 for SP-I and 0.3 6 mAf for SP-II were obtained. Saturation 

 curves and double-reciprocal plots were determined for both 

 proteins (data not shown) when assayed in the forward 

 direction with DHS and NADPH. SP-I had a Km of 0.3 6 mAf with 

 DHS and SP-II had a difference of 0.26 mAf for DHS. The Km 

 value for NADPH was the same for SP-I and SP-II at 0.01 mAf. 

 SP-I and SP-II had different Km values for DQT activity with 

 substrate DHQ (0.07 and 0.04 mM DHQ, respectively, data not 

 shown) . Table 5-1 relates the Km and Vmax values and the 

 ratio of Vmax to Km for each functional domain activity of 

 the two proteins. 



