Characterization of pH-dependent structural properties of hydrolase PncA using NMR

Abstract

Catalytic enzyme Pyrazinamidase (PncA) from Mycobacterium tuberculosis can hydrolyze substrate pyrazinamide (PZA) to pyrazoic acid (POA) as active form of compound. Using NMR spectroscopy, pH-dependent catalytic properties were monitored including metal binding mode during converting PZA to POA. There seems to be a conformational change through zinc binding in active site from the perturbation of peak intensities in series of 2D HSQC spectra the conformation changes through zinc binding.

Figure 1. 2D HSQC spectra for pH titration of PncA. 2D HSQC spectra were plotted in various pH (colored by black (pH 5.3), red (pH 5.6), green (pH 5.9), blue (pH 6.1) and magenta (pH 6.7).

Figure 2. 2D HSQC spectra of zinc titration of PncA. One equivalent of zinc (red) was added to native PncA sample in pH 6.7 (black).

Figure 3. Analysis of ¹H-¹⁵N HSQC spectra of PncA with and without zinc ion at 313 K, pH 6.7. Chemical shift differences (top) for all clearly detectable peaks and relative peak intensity (bottom) of two HSQC spectra with and without zinc ion were shown.

Figure 4. Active site comparison of PncA. Active site residues of P. horikoshii PncA was colored by orange and that of M. tuberculosis by yellow. Histidine 57 was colored by red, substrate PZA by cyan and zinc ion by blue.