Steady-state and transient kinetic analysis of the inhibition of PDF_Ec activity by AMT in the presence of fixed and varied concentrations of substrate. Panel A depicts the inhibition of PDF_Ec activity by 500 µM AMT as a function of increasing incubation time of the inhibitor in DMF. The reaction progress curves of the PDF_Ec-catalyzed reaction are shown in the absence (solid circles) and presence of 500 µM AMT incubated in DMF for 30 min (open circles) and 7 days (open triangles). The solid lines represent the linear fit of the initial portion of the reaction progress curves, whereas the dashed line represents the best fit of the data by Eq. 1. Panel B shows the dependence of k_obs (open circles) on the concentration of DMF-activated AMT and represent the average k_obs value from two independent assays. The dashed line represents the best fit of the data by Eq. 2, which provided values of 6.4 × 10⁻⁴ and 1.3 × 10⁻⁴ sec⁻¹ for k₃ and k₄, respectively, and a K_i value of 190 µM. The solid line represents the best fit of the data by Eq. 3 (with the value for K_i^* fixed at 3.7 µM) and provided values of 7.3 × 10⁻⁴ and 2.2 × 10⁻⁵ sec⁻¹ for k₃ and k₄, respectively, as well as a K_i value of 129 µM. The inset of panel B shows the reaction progress curves for the PDF_Ec-catalyzed reaction in the presence of 150 µM (open circles), 300 µM (solid squares), 600 µM (open squares) and 1200 µM (solid triangles) inhibitor with the solid lines depicting the best fit of the data by Eq. 1. Panel C shows the dependence of k_obs for the PDF_Ec-catalyzed reaction in the presence of 250 µM DMF-activated AMT as a function of ^FMet-Leu-p-nitroanilide concentration. The solid line indicates the best fit of the data by Eq. 4 which provided a value of 0.0027 sec⁻¹ for k₀ and a K_m of 83 µM. The double-reciprocal plot for the PDF_Ec-catalyzed reaction is given in the inset of panel C at concentrations of 0, 30, 50 and 100 µM AMT (open squares, solid squares, open triangles and solid circles, respectively). The solid lines of the inset represent the simultaneous fit of all the datasets by Eq. 5, which provided values of V_max and K_m of 0.65 µM sec⁻¹ and 54 µM, respectively, as well as a K_i of 170 µM.

Mass spectral analysis of AMT upon incubation in DMF. Panel A shows the mass spectrum of AMT (in units of ion current) after 15 days incubation in DMF, with the mass to charge ratio (m/z) indicated by the numbers located above each peak. The inset of Panel A indicates the fragmentation spectrum of the m/z 265 species determined via MS² analysis. Panel B shows the intensity of the m/z 265 peak as a function of incubation time of AMT in DMF.

Representations of bis-AMT and ^FMet-Leu-p-nitroanilide in complex with the active site of PDF_Ec. Panel A depicts the stereo representation of bis-AMT docked to the active site of PDF_Ec. Docking of bis-AMT to the structure of the Ni²⁺-substituted form of PDF_Ec (PDB ID: 1G2A) [28] was performed on a Microsoft Windows^®-based molecular modeling workstation using Autodock 4.0 [46, 47]. The figure was produced using the UCSF Chimera modeling package [49] and shows the best-docked (lowest energy) structure of bis-AMT (van der Waals surface shown in grid format) as well as active site residues of PDF_Ec which were located within 3.5 Å of the inhibitor. The purple lines indicate coordination bonds whereas the cyan lines indicate putative hydrogen bonds. Panels B and C depict the schematic representation of the putative “subsites” and relevant hydrogen bonding interactions within the active site pocket of PDF_Ec with either ^FMet-Leu-p-nitroanilide or bis-AMT bound (Panels B and C, respectively) [17, 29–32]. In both panels, the bound ligand is shown in black, with hydrogen bonds given by thin dashed lines and coordination bonds given by wide dashed lines.

Chemical structures of 2-amino-5-mercapto-1,3,4-thiadiazole (AMT) and bis-AMT in Panels A and B, respectively.

Time dependent changes in the UV/Vis absorption spectrum and k_obs of AMT as a function of incubation time in DMF. Panel A shows the spectrum of AMT in 45 mM phosphate buffer, pH 7.0 containing 10% glycerol (final DMF concentration = 5%) after different times of incubation in DMF, with the arrows indicating the time dependent changes in the spectrum. Panel B depicts the dependence of the absorption at 350 nm as a function of incubation time in DMF and represents the average of two independent measurements. Panel C shows is the dependence of the k_obs value for the inhibition of PDF_Ec by 500 µM AMT (calculated from the average of two independent measurements at corresponding time-points) as a function of incubation time in DMF. Panel D depicts the correlation between the absorption at 350 nm and the k_obs at corresponding time-points.

UV/Vis absorption spectrum and inhibition properties of bis-AMT. Panel A shows the overlay of the monomeric and disulfide forms of AMT at 250 µM in DMSO (solid and dashed lines, respectively). Panel B depicts the transient-kinetic analysis of the slow-binding inhibition of PDF_Ec by bis-AMT, where the open circles depict the dependence of k_obs on the concentration of inhibitor (as determined by the best fit of the activity traces by Eq. 1). The solid line represents the best fit of the data by Eq. 2, which provided values of 8.0 × 10⁻⁴ and 3.6 × 10⁻⁶ sec⁻¹ for k₃ and k₄, respectively, and a K_i value of 490 µM.

Proposed mechanism for the DMF-mediated activation of AMT to bis-AMT.