Mechanistic Investigation of Bacterial 3-Mercaptopyruvate Sulfurtransferase (3MST) and Design of Inhibitors

Abstract

Hydrogen sulfide (H2S) gas has been found to have a cytoprotective role in bacteria and is also linked with many important physiological functions in humans. We are exploring the mechanism of H2S production by 3-mercaptopyruvate sulfurtransferase (3MST) from Escherichia coli using unnatural substrates comparing it with the mechanism reported for 3-mercaptopyruvate, the natural substrate of the enzyme. Our lab has previously developed these molecules that can produce varying amounts of H2S in the in-vitro reaction with wild-type E. coli 3MST. The structure of these substrates differs vastly from 3-mercaptopyruvate – a non-polar aromatic group has been substituted in place of the highly polar carboxyl group of 3-mercaptopyruvate. Arginine residues in the active-site (Arg 179 and Arg 188) of the enzyme have been reported to hold the substrate in the active-site by electrostatic interactions with the carboxyl and carbonyl groups of 3- mercaptopyruvate. In this study, we investigate the mechanistic details of the turnover of these unnatural substrates by the Ec3MST enzyme by the site-directed mutagenesis of these arginine residues and quantifying the amount of H2S produced in the enzymatic reaction by the Methylene Blue assay. We also computationally dock the unnatural substrates in the active-site of the wild-type Ec3MST enzyme to visualize the interactions with the active-site residues. Lastly, we clone and purify the human 3MST enzyme and characterize it using the unnatural substrates. In conclusion, we establish that a new -cation interaction is responsible for stabilizing the aromatic group of the unnatural substrates in the active-site, compensating for the loss of the electrostatic interaction with the carboxyl group of 3-mercaptopyruvate. Moreover, the mutation of the Arg 179 to a leucine residue increases the activity of the enzyme significantly, which is not observed in the R188L Ec3MST mutation. We also conclude that although human 3MST enzyme has similar active-site residues as the Ec3MST, the amount of H2S produced in the reaction of human 3MST and the unnatural substrates is significantly less as compared to Ec3MST.