Enzyme kinetics

We examined the influence of photomodulation on enzyme kinetics by measuring the activities of PTP1B_PS and PTP1B_PS* on pNPP in the presence and absence of light (i.e., we used dark and light plates as described above). Briefly, we prepared 100-μL reactions consisting of buffer (50 mM HEPES, 0.5 mM TCEP, pH 7.5), substrate (0.2, 0.5, 1, 2.5, 5, 10, and 15 mM pNPP), and enzyme (25 nM); at 4, 8, 12, 16, and 20 min after initiating the reaction, we measured the production of p-nitrophenol (405_abs) on a SpectraMax M2 plate reader; and we used DataGraph to fit initial rates to a Michaelis–Menten model of enzyme kinetics. Final values of k_cat and K_m reflect the mean of independent estimates determined from three Michaelis–Menten curves; error k_cat and K_m reflects the standard error of those estimates.

We examined the inhibitory effect of DPM-1001 on PTP1B-mediated hydrolysis of pNPP as follows: (i) We carried out the aforementioned pNPP reactions in the presence of different concentrations of DPM-1001 (0, 20, 40, 60 μM for PTP1B_1-405 and PTP1B_PS*; 0, 100, 200, 400 μM for PTP1B₃₂₁ and PTP1B_PS). (ii) We used MATLAB’s “nlinfit” and “fminsearch” functions to fit (a) initial-rate measurements collected in the absence of inhibitors to a Michaelis–Menten model and (b) initial-rate measurements collected in the presence and absence of inhibitors to four models of inhibition (i.e., competitive, noncompetitive, uncompetitive, and mixed inhibition⁶³). (iii) We used an F-test to compare the fits to (a) a mixed model, which has two parameters, and (b) each nested single-parameter model with the lowest sum of squared errors for a given dataset. DPM-1001 exhibited mixed inhibition for all constructs (p < 0.01, two-tailed Student’s t test). (iv) We estimated IC₅₀’s by using the best-fit kinetic model to determine the inhibitor concentration required to reduce initial rates by 50% on 15 mM pNPP. This high substrate concentration minimizes the concentration dependence of IC₅₀’s. We used the MATLAB function “nlparci” to determine the confidence intervals of kinetic parameters and propagated those intervals to estimate the corresponding confidence on IC₅₀’s.

We compared the activities of PTP1B_1-281 and PTP1B_1-321 on pNPP by using a continuous assay. Briefly, we prepared 100-μL reactions consisting of buffer (50 mM HEPES, 0.5 mM TCEP, pH 7.5), pNPP (0.2, 0.5, 1, 2.5, 5, 10, and 15 mM), and enzyme (25 nM); we measured the production of p-nitrophenol at 5-s intervals for 270 s (SpectraMax M2 plate reader); we used DataGraph to fit initial rates to a Michaelis–Menten model. Final values of k_cat and K_m reflect the mean of independent estimates determined from three Michaelis–Menten curves; error k_cat and K_m reflects the standard error of those estimates.

Finally, we evaluated the reversibility of our LOV2-based light switch by illuminating 25 μM of PTP1B_PS (50 mM HEPES, 0.5 mM TCEP, pH 7.5) for 10 s and by, subsequently, monitoring its activity on 5 mM pNPP after 5 min in the dark. To minimize error, we repeated this experiment three times with seven cycles per experiment (Supplementary Fig. ²).