Figure 5. Non-covalent tethering enhances the rate of phosphorylation and depends on the relationship between effective molarity and K_D.

(A) Schematic of a model scaffold with a non-covalent tether that recruits a substrate to a kinase. The kinase is fused to SYNZIP6 (PKA-SYNZIP6) and the substrate is fused to SYNZIP5, which interact to form the tethered complex. In the corresponding free reaction, we used a kinase without SYNZIP6.

(B) Plot of k_obs vs. [substrate] for the tethered and free reactions for [substrate] < 0.06 μM. At concentrations below the effective molarity, the values of k_obs for the tethered reaction are significantly larger than the untethered reaction. The solid red line is a fit to a kinetic model (Scheme 1 and Supporting Information) that includes a contribution from k_intra, which accurately fits the tethered reaction data. The dotted lines are fits to a model where k_obs = (k_cat/K_M)[S] (this linear fit forces the line through zero). Both fits include data at higher substrate concentrations (full dataset is shown in panel C), and for the tethered reaction this linear model fails to account for the observed rate increase at low substrate concentrations. For the untethered reaction, the data scale linearly with [substrate]. The reaction of PKA-SYNZIP6 is also first order in enzyme concentration (Figure S6).

(C) Plot of k_obs vs. [substrate] over an expanded range of substrate concentrations up to 0.25 μM. The dotted lines are fits to a simple linear model (k_obs = k_cat/K_M [S]), and the solid red line is a fit to the complete model that includes k_intra (Scheme 1 and Supporting Information). For the untethered reaction, the slope corresponds to a bimolecular rate constant (k_cat/K_M) of 4.2 × 10⁴ M⁻¹ s⁻¹. For the tethered reaction, the value of k_cat/K_M obtained from the fit to the complete model (solid red line) is 5.6 × 10⁴ M⁻¹ s⁻¹. This value differs from the untethered reaction by a factor of 1.3, likely due to prep-to-prep variations in the total activity of PKA-SYNZIP6 versus free PKA.

(D) Plot of k_obs vs. [substrate] for the tethered reaction with the SYNZIP 5/6 pair and with two SYNZIP5 truncations that weaken the binding affinity (increasing K_D) (Figure S19). At [substrate] < 0.03 μM, values of k_obs decrease as K_D increases. At [substrate] = 0.25 μM, well above the effective molarity, values of k_obs are indistinguishable for substrates with different SYNZIP interaction affinities. The solid lines represent kinetic models using the values of k_intra and k_cat/K_M from the untruncated pair in Figure 5C, and the corresponding K_D value from each truncated pair. For SYNZIP5trunc2, we used a K_D of 50 μM (using larger K_D values does not change the prediction, see Figure S18B & C). Each [product] vs time trace was measured from separate reactions in duplicate. Error bars in B-D represent the standard error for k_obs obtained from a linear fit to [product] vs time for both datasets.