Table V.

Activation by 3-PGA of the synthesis of ADP-Glc

E. coli AC70R1-504 cells were cotransformed with two plasmids. Plasmid pMON17336 encodes either the wild-type (wt) large subunit of the potato tuber ADP-Glc PPase or a mutated large subunit. The other plasmids (pMLaugh10) encodes either the wild-type small subunit or a single amino acid mutant. Enzymes were expressed and purified as described in Methods. Specific activities of the mutants were determined at saturated concentrations of activator (3-PGA) and substrates (1.5 mm ATP, 0.5 mm Glc-1-P, 7 mm Mg²⁺). In the case of mutants L_wtS_K404A and L_K417ES_441E, concentrations of substrates were increased four times but still no activation was observed. Every constant was determined at least twice and the difference was <10% of the average in all cases. Average values are shown. Inhibition has been observed at higher concentrations of 3-PGA.

Subunit			A0.5		Specific Activity	Activationa
pMON17336 (Large)		pMLaugh10 (Small)	3-PGA	Ratio mutant/wt
			mm		units mg−1	−fold
		wt	3.5		32	>100
wt	+	wt	0.10	1	30	30
K455A	+	wt	0.22	2	22	>100
K455E	+	wt	0.77	8	16	>100
K417A	+	wt	0.30	3	11	25
K417E	+	wt	1.30	13	5	>30
wt	+	K441A	3.2	32	16	>30
wt	+	K441E	8.3	83	3	>30
wt	+	K404A	No activation		0.26	None
K417A	+	K441A	6.0	60	9	31
K417E	+	K441E	No activation		0.14b	None

^aRatio between the activity at saturated concentrations and in the absence of 3-PGA. 

^bIn this case, specific activity was determined in the absence of 3-PGA. On the other hand, specific activity is estimated to be higher because the purity reached was about 40%. One unit is defined as 1 μmol of ATP formed in 1 min.