103 



Table 5-1. Kinetic parameters for SP-I and SP-II 



Enzyme 



Substrate 



Km (mAf) Vmax (nmol/min) 



(Vmax/Km) 



SP-I SDH 



SHK 



0.80 



0.60 



0.75 





NADP 



0.02 



0.59 



29.50 





DHS 



0.36 



0.25 



0.69 





NADPH 



0.01 



0.26 



26.00 



QDT 



DQH 



0.07 



1.96 



28.00 



SP-II SDH 



SHK 



0.36 



0.60 



1.66 





NADP 



0.02 



0.59 



29.50 





DHS 



0.26 



0.27 



1.04 





NADPH 



0.01 



0.26 



26.00 



QDT 



DQH 



0.04 



1.47 



36.00 



Inhibitor effects on enzyme activities 



Protocatechuic acid (0.5 mM) inhibited SDH activity of 

 both SP-I and SP-II at about 15% at saturating substrate 

 concentrations (5.0 mM SHK and 1.0 mAT NADP^) and about 57% 

 for each at 0.8 mM SHK (SP-I), 0.36 mW SHK (SP-II) and 0.2 

 mM NADP*. PCA did not inhibit the DQT activities of the two 

 proteins. PCMB was inhibitory to SDH activities for both 

 functional domains and activated the DQT functional domains 

 from both bifunctional proteins as shown in Table 5-2. 



Neither SP-I or SP-II were effected by additions of 0.5 

 mAf PHE, TYR, TRP, quinate, and cinnamic acid when added to 

 saturated substrate reaction mixes and assayed for DQT or 

 SDH activities. Metals at 0.2 mM concentrations (calcium, 

 magnesium, manganese, cobalt, zinc, and iron) had no effect 

 on activities of either protein. Incubation of DTT had no 

 effect on any of the activities. EDTA at 10 and 125 vnM had 



