24 

 were included in the reaction sample of 100 /xl that were 

 incubated at 32°C for 3 min. Controls included samples 

 without AGN or enzyme. 

 Dehydroquinase 



Dehydroquinase activity was assayed spectro- 

 photometrically at 234 nm at room temperature (24°C) by 

 following the rate of increase of product, dehydroshikimate, 

 when dehydroquinate was provided as substrate . Unless 

 otherwise stated, a reaction sample included 0.5 mAT DHQ, 

 enzyme and 50 mM EPPS-KOH buffer at pH 8 . 6 . An extinction 

 coefficient of 12,000 was used to calculate activity (37). 

 DQT was also assayed f luorometrically via a coupled assay 

 with SDH. DHS formed from DHQ by the dehydratase reaction 

 was reduced with NADPH (cof actor for SDH) and the rate of 

 NADPH disappearance was followed. Reaction samples 

 contained . 5 mM DHQ, . 2 mAf NADPH in 50 mM EPPS-KOH at pH 

 8.6 for column fractions or 50 mM Bis-tris propane buffer, 

 pH 7.5, for other studies as stated. Controls were carried 

 out for each enzyme assayed by adding only one substrate for 

 about three to five min before adding the second substrate. 

 Dehydrogenases 



Dehydrogenase reactions were usually assayed 

 fluorometrically at room temperature (24°C) with cofactor 

 NAD(P)* for 1 to 2 min before addition of substrate, and the 

 rate of formation of NAD(P)H was monitored (340 nm emission 



