Figure 1.
Structure of site I in PGDH (PDB code: 1YBA).
Site I is represented by the green surface, the active site is indicated by orange spheres, and the cofactor NAD+ and the endogenous allosteric L-serine are illustrated in stick and sphere, respectively.
Figure 2.
Structures of the compounds 1-3.
The SPECS IDs of compounds 1-3 are AN-698/40677526, AN-023/41981714 and AG-205/07681005.
Figure 3.
Dose-response curves of compounds 1-3.
(A) Residual enzyme activity versus compound concentration. The IC50 values were 34.8±1.3 for compound 1, and 58.0±9.0 µM for compound 2. The AC50 value was 34.7±4.5 nM for compound 2. (B) Enzyme inhibition dose-response curves of compound 3. The fitted IC50 value was 131±12 µM. (C–D) SPR dose-response curves of compound 1, compound 2 with immobilized PGDH, respectively. The KD values were 42.6±2.1 for compound 1 (C), and 19.0±1.9 µM for compound 2 (D). KD values of the compounds 1 and 2 were obtained by fitting the data sets to 1:1 Langmuir binding model using Biacore T200 Evaluation Software. Residuals for all SPR sensorgrams were less than 2 RU. Chi-square values were 2.4 for C and 1.2 for D.
Figure 4.
Competitive assay of compounds 1-3 with the substrate.
(A) Compounds 1 and 2 and the substrate do not competitively bind to the same site. Increasing the substrate concentration led to higher inhibition rates of the compounds in contrast to lowered inhibition as expected for competitive inhibitors, indicating that these compounds do not bind to the substrate-binding site. (B) Substrate competition curve of compound 3. The percentage inhibition did not change along with the increase of substrate concentration, indicating that there are no significant interactions between compound 3 and the substrate binding site.
Figure 5.
(A–B) The complex structure model of compounds 1 and 2 binding to site I in PGDH. Compound 1 (A), compound 2 (B), and the mutation sites are shown in stick representation, while site I is in surface representation. (C–D) SPR direct binding curves of WT, Y410A, F147A and E129AK256A. WT PGDH and its mutants were immobilized on the sensor chip. Compounds 1 (C) and 2 (D) were injected over the chip at a fixed concentration of 50 and 25 µM, respectively.
Table 1.
Mutation effects on the inhibition rates of compounds.
Figure 6.
The Km, kcat and kcat/Km values versus compound concentration.
The kinetic data for PGDH show that the values of Km decrease with increasing concentrations of 1 and 2 ranging from 0 µM to 120 µΜ, while values of kcat and kcat/Km show an initial increase and then a decrease.