FIGURE 8.

Rates of l-aspartate binding to Glt_Ph F273W in the presence of 1 m NaCl. The top panels show the stopped-flow fluorescence measurements. Each trace (black) represents an average of 6–10 individual traces. A, 1.5 μm F273W was mixed with varying l-Asp concentrations as indicated, and individual traces were fitted with a double exponential equation (gray). In the bottom panel the k_obs values resulting from the fits with assumed pseudo-first-order reaction are plotted against the corresponding aspartate concentrations. The slope of the linear fit gives the k_on, and the intercept with the k_obs axis gives the k_off value. The inset shows the data at low aspartate concentrations. B, 1.5 μm F273W was mixed with 100 μm l-asp at different temperatures as indicated, and individual traces were fitted with a single exponential equation. The bottom panel shows the Arrhenius plot of the derived k_obs. The gray line demonstrates the linear fit of the data according to the Arrhenius equation ln(k) = B + (ln(E_a)/RT), with E_a representing the activation energy, B representing the frequency factor, T representing the absolute temperature, and R representing the gas constant. The linear fit results in an E_a = 42.6 kJ mol⁻¹.