Table III.

Inhibition of the ADP-Glc PPase mutants by phosphate in the pyrophosphorolysis direction

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 plasmid (pMLaugh10) encodes either the wild-type small subunit or a single amino acid mutant. Enzymes were expressed and purified as described in Methods. The substrates and cofactors were 2 mm ADPGlc, 1.5 mm PPi, and 7 mm Mg²⁺. The I_0.5 of Pi was determined as described in Methods. Every constant was determined at least twice and the difference was <10% of the average in all cases. Average values are shown. The 3-PGA activation curve was performed in presence of 0.5 mm phosphate.

Subunit			I0.5		A0.5
pMON17336 (Large)		pMLaugh10 (Small)	Phosphate	Ratio mutant/wt	3-PGA activation +0.5 mm Pi	Ratio +Pi/−Pia
			μm		μm
wt	+	wt	74	1.0	56	25
K455A	+	wt	19	0.25	160	27
K455E	+	wt	20	0.25	280	28
K417A	+	wt	76	1.0	400	20
K417E	+	wt	22	0.3	600	22
wt	+	K441A	32,000	432	240	2.0
wt	+	K441E	16,000	216	800	1.9
wt	+	K404A	48,000	650	8000	1.2

^aTo determine the ratio A_0.5(+Pi)/A_0.5(−Pi), the A_0.5 value in the absence of Pi was taken from Table II.