Table 3 Dissociation constants of the WT PNP and the Y160W mutant complexes with phosphate (Pi), and the WT PNP/Pi and the Y160W mutant/Pi complexes with formycin A (FA), determined by various experimental methods.

From: Single tryptophan Y160W mutant of homooligomeric E. coli purine nucleoside phosphorylase implies that dimers forming the hexamer are functionally not equivalent

Enzyme

Titrant

 

Fluorescence titrations

Thermophoretic titrations

CD titrations

Kd ± SD

Confidence intervals

Kd ± SD

Confidence intervals

Kd ± SD

Confidence intervals

WT

Pi

Kd1

(4.4 ± 1.1) μM

(1.8–6.6 ) μM

(7.0 ± 3.4) μM

(2.9–12.9) μM

(0.58 ± 0.80) μM

(< 0.07–2.12)* μM

Kd2

(84 ± 12) μM

(59–118) μM

  

(107 ± 30) μM

(76–144) μM

Kd3

(1.77 ± 0.14) mM

(1.47–2.23) mM

(1.64 ± 0.14) mM

(1.38–1.94) mM

(1.03 ± 0.29) mM

(0.76–1.46) mM

Y160W

Pi

Kd1

(7.0 ± 1.4) μM

(4.8–10.9) μM

(3.20 ± 3.06) μM

(0.4–15.4) μM

(4.13 ± 3.77) μM

(0.3–9.7) μM

Kd2

(200 ± 30) μM

(179–409) μM

  

(128 ± 28) μM

(92–177) μM

Kd3

(3.2 ± 0.6) mM

(2.6–5.2) mM

(1.282 ± 0.079) mM

(1.12–1.45) mM

(2.33 ± 0.17) mM

(2.05–2.74) mM

Enzyme

Titrant

 

Fluorescence titrations

Thermophoretic titrations

Calorimetric titrations

Kd ± SD

Confidence interval

Kd ± SD

Confidence interval

Kd ± SD

Confidence interval

WT/Pi

FA

Kd1

#

#

(0.0107 ± 0.0074) μM

(0.0005–0.0334) μM

(8.9 ± 1.9) μM

(5.5–10.8) μM

Kd2

#

#

  

(55 ± 19) μM

(40–81) μM

Kd3

#

#

(186 ± 38) μM

(117–303) μM

(812 ± 631) μM

(321–1615) μM

Y160W/ Pi

FA

Kd1

#

#

(0.137 ± 0.027) μM

(0.092–0.201) μM

(6.75 ± 0.30) μM

(4.01–10.10) μM

Kd2

#

#

(110 ± 23) μM

(29–246) μM

(148.9 ± 9.8) μM

(97.0–216.3) μM

Kd3

#

#

(1754 ± 774) μM

(994–5324) μM

(655 ± 37) μM

(412–1033) μM

  1. Experiments were conducted at 25 °C, in 50 mM Tris/HCl buffer at pH 7.6, and in the case of titrations with formycin A in the presence of 50 mM phosphate buffer pH 7.6. Dissociation constants obtained from the global fitting (see “Methods” section) with standard deviations are shown, and also the confidence intervals at 3σ level (in parenthesis) .
  2. *In this case the confidence interval is not closed from the bottom, as the lower bound is lower than the lowest ligand concentration used in titrations (0.007 μM).
  3. #The strong absorption of FA makes fluorimetric titrations impossible even at moderate ligand concentrations.
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