Table 2 Kinetic and equilibrium (fluorescence) data for the p53-peptides and MDM2 binding reactiona.

From: Kinetic and thermodynamic effects of phosphorylation on p53 binding to MDM2

p53-peptide

kon 106 (M−1 s−1)

koff (s−1)

Kd (koff/kon) (μM)c

Kd (μM)d

Ac-ETFSDLWKLLPEN-NH2 (P53-WT)c

1.6 ± 0.1

9.0 ± 0.3

5.5 ± 0.5

3.0 ± 1.2

Ac-EeFSDLWKLLPEN-NH2 (P53-E18)

3.8 ± 0.7

16 ± 2

4.2 ± 0.9

2.3 ± 0.6

Ac-ETFdDLWKLLPEN-NH2 (P53-D20)

9.5 ± 0.9

7 ± 2

0.7 ± 0.2

1.5 ± 0.4

Ac-EeFdDLWKLLPEN-NH2 (P53-E18-D20)

4 ± 2

18 ± 6

4.0 ± 3.0

8.3 ± 4.9

Ac-EeFdDLWeeLPEN-NH2 (P53-E18-D20-E24-E25)b

    

Ac-EeFeDLWKLLPEN-NH2 (P53-E18-E20)

2.7 ± 0.9

17 ± 2

6 ± 2

 

Ac-EdFSDLWKLLPEN-NH2 (P53-D18)

5 ± 3

23 ± 9

5.0 ± 3.0

 

Ac-ETFeDLWKLLPEN-NH2 (P53-E20)

9 ± 2

8 ± 5

0.9 ± 0.6

0.7 ± 0.1

Ac-EdFeDLWKLLPEN-NH2 (P53-D18-E20)

8 ± 2

13 ± 4

1.6 ± 0.6

3.2 ± 0.9

Ac-EdFdDLWKLLPEN-NH2 (P53-D18-D20)

11 ± 4

20 ± 11

1.8 ± 1.2

 

Ac-ETFpsDLWKLLPEN-NH2 (P53-pS)

3.3 ± 0.8

5 ± 1

1.6 ± 0.6

0.3 ± 0.1

Ac-EptFSDLWKLLPEN-NH2 (P53-pT)

2.8 ± 1.6

20 ± 3

7.0 ± 4.0

3.0 ± 1.6

Ac-EptFpsDLWKLLPEN-NH2 (P53-pTpS)

1.9 ± 1.1

11 ± 2

5.7 ± 3.5

2.5 ± 0.9

  1. aExperiments were carried out at 15 °C. Data errors are fitting errors from the slope and the y-axis intercept of the pseudo-first-order plots. Mutations are indicated in bold, in lower case lettering. Final concentrations of the peptides into the stopped-flow chamber were 0.125 μM.
  2. bNo exponential behaviour was observed in any trace at any of the concentrations of MDM2 tested.
  3. cDetermined assuming a two-state binding reaction without any intermediates.
  4. dDetermined from fluorescence titrations. The change in fluorescence at 315 nm (after excitation at 280 nm) as a function of p53-peptide concentration was fitted to Eq. (1).
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