Book of Abstracts: Albany 2007
June 19-23 2007
Saturation Transfer Difference NMR Studies on the Binding of Pyrazinamide and 5-Chloropyrazinamide to Mycobacterium tuberculosis Fatty Acid Synthase I
We have previously shown that an analog of pyrazinamide (PZA), 5-chloropyrazinamide (5-Cl-PZA) inhibits fatty acid synthase I (FASI) in Mycobacterium tuberculosis (Mtb). FASI has been purified from Mycobacterium smegmatis 2700 mc2, a recombinant strain where the native fas1 gene has been deleted and replaced with Mtb fas1 gene. Following purification, FASI enzymatic activity was measured using a spectrometric assay, which monitors NADPH oxidation. Both 5-Cl-PZA and PZA showed concentration and substrate dependence consistent with competitive inhibition of FASI. These results were validated by a radiolabeled fatty acid assay.
To further prove that 5-Cl-PZA and PZA bind to FASI, we used saturation transfer difference (STD) NMR experiment. In this experiment, binding of the ligand (PZA or 5-Cl-PZA) to the target (FASI) is measured by the degree of ligand NMR signal saturation.
NMR results clearly show that both 5-Cl-PZA and PZA bind to FASI. Based on STD competition titration, 5-Cl-PZA binds to FASI with dissociation binding constant KD of 90 μM, which is significantly lower than the PZA binding constant KI of 250 μM. These results were in agreement with data obtained by spectrometric assays.
These NMR experiments are the first step in systematic structural analysis of the binding of PZA and its analogs to FASI.
H. Sayahi1, *
1Department of Chemistry