Figure 7.

Replots of the kinetic parameters obtained from fitting individual time courses at each substrate concentration to eqs 8–10 in ${\text{H}}_2\text{O}$ (blue) and ${\text{D}}_2\text{O}$ (red). Substrate concentrations used to determine the transient rate constants were 1.5–79 μM, compared to substrate concentrations used for the steady-state rate and burst amplitudes (0.5–8.75 μM). (a) Fitting of the steady-state rate vs [Z-FR-AMC] (eq 1) resulted in the following values: $k_{\text{cat}}=34.2\pm 1.9{\text{s}}^{-1}$ and $K_{\text{a}}=3.9\pm 0.5\mu \text{M}$ in ${\text{H}}_2\text{O}$, and $k_{\text{cat }}=14.6\pm 0.3{\text{s}}^{-1}$ and $K_{\text{a}}=1.4\pm 0.1\mu \text{M}$ in ${\text{D}}_2\text{O}$. (b) Fitting of the burst amplitude vs [Z-FR-AMC] (eq 9) resulted in the following values: $k_{\text{cat }}=34{\text{s}}^{-1},K_{\text{a}}=1.1\pm 0.1\mu M$, and $k_{\text{dac}}=36.5\pm 0.8{\text{s}}^{-1}\left({\text{H}}_2\text{O}\right)$, and $k_{\text{cat}}=14.6{\text{s}}^{-1},K_{\text{a}}=0.6\pm 0.06\mu \text{M}$, and $k_{\text{dac}}=14.4\pm 0.2{\text{s}}^{-1}\left({\text{D}}_2\text{O}\right)$. (c) Fitting of the transient rate constant vs [Z-FR-AMC] (eq 10) resulted in the following values: $k_{\text{ac}}=233\pm 3{\text{s}}^{-1},k_{\text{dac}}=12\pm 3{\text{s}}^{-1}$, and $K_{\text{ia}}=9.8\pm 0.6\mu \text{M}\left({\text{H}}_2\text{O}\right)$, and $k_{\text{ac}}=167\pm 3{\text{s}}^{-1},k_{\text{dac}}=14\pm 3{\text{s}}^{-1}$, and $K_{\text{ia}}=9.0\pm 0.7\mu \text{M}\left({\text{D}}_2\text{O}\right)$.