Table II.

Activation by 3-PGA in the pyrophosphorolysis reaction

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. Specific activities of the mutants were determined at saturating concentrations of activator (3-PGA) and substrates. Every constant was determined at least twice and the difference was <10% of the average in all cases. Average values are shown.

Subunit			A0.5		Specific Activity	Activationa
pMON17336 (Large)		pMLaugh10 (Small)	3-PGA	Ratio mutant/wt
			μm		units mg−1	−fold
		wt	900		55	>500
wt	+	wt	2.2	1	48	3.6
K455A	+	wt	6	3	47	6
K455E	+	wt	10	5	51	50
K417A	+	wt	20	9	13	22
K417E	+	wt	27	12	14	18
wt	+	K441A	120	54	32	2.9
wt	+	K441E	420	191	39	3.1
wt	+	K404A	6800	3090	12	130
K417A	+	K441A	390	177	7	80
K417E	+	K441E	6500	2955	5b	96

^aRatio between the activity at saturated concentrations and in the absence of 3-PGA. In the case of the wild-type small subunit, the activity in the absence of activator was negligible even when 0.5 μg of purified enzyme was added in the reaction mixture. 

^bSpecific activity is estimated to be higher because the purity was about 40%. One unit is defined as 1 μmol of ATP formed in 1 min.