Fig. 5.

Substrate inhibition of AKR1D1 by testosterone. Velocity versus substrate plot for AKR1D1 showing substrate inhibition using testosterone. Assays contained 0.078 μM enzyme, 1–40 μM testosterone, 12 μM NADPH, 4% acetonitrile in 100 mM potassium phosphate buffer, pH 6.0, in a final volume of 1 mL. Reactions were monitored fluorometrically by using an excitation wavelength of 340 nm (slit-width 5 nm) and an emission wavelength set at 450 nm (slit-width 10 nm) at 37 °C. Kinetic analysis of initial velocities obtained was performed using the Henri-Michaelis-Menten equation for uncompetitive substrate inhibition v = (V_max × [S])/(K_m + [S] + [S]²/K_i) and fit using the program GraphFit. The iterative fits provided estimates of V_max, K_m and K_i and associated standard errors. This research was originally published in the Journal of Biological Chemistry. Di Costanzo et al., (2008).