TABLE 3.

Effect of varying pH and temperature on activity of PK-WT and mutant enzymes

Protein	Optimum pH		Slope (na)		Slope (ni)
	-Fru-1,6-P2	+Fru-1,6-P2	-Fru-1,6-P2	+Fru-1,6-P2	-Fru-1,6-P2	+Fru-1,6-P2
A. pH-dependent studya,b
PK-WT	7.4 ± 0.2	7.4 ± 0.2	3.68 ± 0.2	3.7 ± 0.12	0.35 ± 0.12	0.34 ± 0.1
K422R	7 ± 0.1	7 ± 0.1	3.59 ± 0.1	3.48 ± 0.1	0.91 ± 0.1	0.25 ± 0.2
H391Y	7 ± 0.2	7 + 0.3	1.56 ± 0.1	1.5 ± 0.12	0.86 ± 0.08	0.16 ± 0.1
Protein	Incubation at 60 °C, activity lost after 10 min			Cooling in ice, activity regained after 15 min			Incubation at 37 °C, activity left after 3 h
		-Fru-1,6-P2	+Fru-1,6-P2	-Fru-1,6-P2	+Fru-1,6-P2	-Fru-1,6-P2	+Fru-1,6-P2
	%			%			%
B. Temperature-dependent studya,b
PK-WT	100	100	0	0	44 ± 2	58 ± 1
K422R	100	100	0	0	20 ± 1	32 ± 2
H391Y	2 ± 0.01	0	64 ± 2	64 ± 3	99 ± 1	100

^aAssays were done under standard assay conditions as described under “Experimental Procedures.”

^bFru-1,6-P₂ was used at a final concentration of 2.5 mm wherever required.