Table 4.

Additivity of mutant cycles

Reference		Modification #1			Modificaiton #2			Double modification
	Ka-PEP [mM]	Modification description	Ka-PEPb [mM]	ΔΔG1a (kcal/mol)	Modification description	Ka-PEP [mM]	ΔΔG2a (kcal/mol)	Calculated ΔΔG1 + ΔΔG2 (kcal/mol)	Ka-PEP [mM]	Δ ΔG3a (kcal/mol)
Wild Type	0.240±0.002	Δ2–10	0.605±0.004	0.55±0.01	C436S	0.802±0.004 c	0.726±0.008	1.28±0.02	1.139±0.003	0.938±0.007
					C436N	0.703±0.004 c	0.647±0.008	1.20±0.02	1.016±0.005	0.869±0.008
		S12D	0.552±0.007	0.50±0.01	C436S	0.802±0.004 c	0.726±0.008	1.23±0.02	0.89±0.01	0.79±0.01
					C436N	0.703±0.004 c	0.647±0.008	1.15±0.02	0.95±0.01	0.83±0.01
					C338A	0.93±0.01	0.82±0.01	1.32±0.02	2.09±0.02	130±0.01.

^aΔG = change in free energy associated with PEP binding. ΔΔG = the difference in ΔG of PEP binding of modified protein relative to that of the wild type enzyme. Conversion of K_a-PEP to ΔG is based on the generally accepted view that PYK isozymes function as rapid equilibrium systems (30). As such, the free energy associated with PEP binding was calculated by ΔG=−RTln K_a-PEP.

^bvalues from (12)

^cvalues from Table 2