FIGURE 6.

Kinetic reaction scheme for the interaction between PSD-95 PDZ1–2 and a dimeric peptide. The initial association and dissociation rate constants (k₁, k₂, k₋₁, k₋₂) in the scheme were obtained from experiments with monomeric peptide, but are fully consistent with the dimeric peptide binding data. The reverse rate constants (k₋₃, k₋₄) were measured from displacement kinetics of the PDZ tandem constructs and dimeric peptide. In the displacement reaction from wild type PDZ1–2 tandem, only the slowest intramolecular k_off can be determined (0.9 s⁻¹); the rate constant of 1.8 s⁻¹ is inferred from, first, the intermolecular k_off values between monomeric peptide and single PDZ domains and, second, from the ratio of intramolecular k_off values from dimeric peptide and Trp mutant tandems PDZ1*–2 and PDZ1–2* (not shown). In both cases the off-rate constant from PDZ1 is roughly two times that of PDZ2. The intramolecular forward rate constant k₃ was determined from the dependence of the slow step for PDZ1–2* and dimeric peptide (Fig. 5F, k_obs^max ≈ k₃ = 62 s⁻¹), as described in the Results section. Finally, k₄ was calculated as 31 s⁻¹, given that the product of rate constants on one half of the scheme must equal that of the other half.