Binding Site of Divalent Transition Metal Cations on Esterase Rv0045c from Mycobacterium tuberculosis

Isobel Bowles Butler University
Faculty Sponsor(s): Geoffrey Hoops Butler University
Tuberculosis is a leading cause of death throughout the world today, caused by the bacterium Mycobaterium tuberculosis. Multiple enzymes, including esterase Rv0045c of M. tuberculosis, aid in the disease’s persistence. I investigated the inhibition of Rv0045c by divalent metals and the structural components of Rv0045c responsible for its regulation. Steady state kinetic assays were run using fluorogenic ester substrates to analyze the effect of divalent metal cations on Rv0045c catalysis. Based on the kinetic data results, diverse divalent metals appear to serve as allosteric inhibitors of Rv0045c, as the KM value remained steady throughout the assays despite the presence of divalent metals, while the kcat value decreased drastically. The following metals inhibited Rv0045c with differing specificities as measured by IC50 values: Copper (Cu2+; 10-5.9M), Zinc (Zn2+; 10-5.6M), Nickel (Ni2+; 10-4.6M), and Cobalt (Co2+; 10-3.5M). Removal of the 6X His-tag by thrombin enzyme failed to remove the inhibitory effect of the divalent metal ions, indicating that the affinity purification tag is not the divalent metal binding site. In an attempt to locate the allosteric binding site of these divalent metal cations, I changed potential amino acid binding residues to alanine via site-directed mutagenesis. The metal inhibition kinetics of wild-type Rv0045c were compared to nine Rv0045c variants. A differing residue with significant potential for binding, was mutated to glutamine rather than alanine, to retain protein structure and function for comparison to WT.
Oral Presentation

When & Where

09:15 AM
Gallahue Hall 101