Table 3.

Nucleotide dissociation constants of WT and mutant F₁

	WT (βY331W)			+ αE284C/γQ274C			+ αE284C/γL276C
MgATP (μM)	Kd1	Kd2	Kd3	Kd1	Kd2	Kd3	Kd1	Kd2	Kd3
Reduction	< 0.01	6.0 (2.5)	34 (17)	0.66 (0.17)	15 (5.5)	93 (44)	< 0.01	10 (3.5)	81 (35)
	R2 = 0.9614			R2 = 0.9755			R2 = 0.9554
Oxidation	< 0.01	7.6 (2.9)	58 (28)	3.0 (0.68)	432 (105)	> 5000#	< 0.01	8.0 (2.2)	140 (56)
	R2 = 0.9628			R2 = 0.9207			R2 = 0.9580
MgADP (μM)	Kd1	Kd2	Kd3	Kd1	Kd2	Kd3	Kd1	Kd2	Kd3
Reduction	0.022 (0.0049)	11 (12)	16 (19)	0.20 (0.041)	19 (5.7)	120 (47)	0.025 (0.0035)	7.3 (1.1)	88 (18)
	R2 = 0.9843			R2 = 0.9859			R2 = 0.9891
Oxidation	0.024 (0.0050)	12 (7.5)	24 (16)	1.3 (0.34)	160 (42)	> 5000#	0.026 (0.0057)	3.6 (0.84)	76 (25)
	R2 = 0.9854			R2 = 0.9408			R2 = 0.9713
MgADP-AlF4 (μM)	Kd1	Kd2	Kd3	Kd1	Kd2	Kd3	Kd1	Kd2	Kd3
Reduction	< 0.01	0.39 (0.13)	12 (4.9)	< 0.01	5.7 (1.2)	42 (11)	< 0.01	3.9 (0.98)	89 (30)
	R2 = 0.9289			R2 = 0.9874			R2 = 0.9665
Oxidation	< 0.01	0.22 (0.072)	27 (11)	0.035 (0.010)	36 (9.9)	> 5000#	< 0.01	2.6 (0.82)	83 (36)
	R2 = 0.9278			R2 = 0.9207			R2 = 0.9375

Nucleotide binding to the catalytic sites was measured at 25 °C by monitoring the fluorescence signal of residue βY331W. Nucleotide dissociation constants (K_d) were calculated by fitting theoretical curves to the experimental data by using a three-site binding model. The coefficient of determination (R²) for each regression is listed. The method applied was unable to resolve K_d values lower than 0.01 μM with confidence. In the underlying titrations, each data point was measured at least three times. The least-square fits to determine binding constants and statistical parameters were based on these individual data points (not on the average of each condition shown in Fig. 1). Standard errors for the K_d values are shown in parentheses.

^#This number was too high to be determined through current experimental method (K_d3 > 5 mM).