Summary:
Hydrogen sulfide (H2S) is the third gasotransmitter after NO and CO and it is produced endogenously via non-enzymatic or enzymatic pathways, with the latter being the most common. Cystathionine beta synthase (CBS), cystathionine gamma lyase (CSE) and 3-mercaptopyruvate sulfurtransferase are the enzymes responsible for its biosynthesis. H2S is involved in a variety of endogenous signaling pathways, thereby regulating various physiological functions. Its contribution to the homeostasis of the human organism becomes particularly evident in cases of either reduced or increased endogenous synthesis, which results in or contributes to different diseases. In order to thoroughly investigate the role of the gaseous transmitter in various pathophysiological conditions, it becomes necessary to elucidate the role of the enzymes of its biosynthesis in mammalian organisms. Given the fact that few bibliographic data is published upon the 3MST enzyme -comparably with the other two enzymes- our thesis focuses on 3MST.
Firstly, we optimized the production of the 3MST enzyme from Escherichia coli bacterial cells. The plasmids that were introduced into the bacterial cells, contained the sequence of human 3MST in frame with glutathione S-transferase (GST). Secondly, following a detailed study of the existing literature and several experiments, we succeeded in optimizing the "methylene blue" method in order to examine the activity of this enzyme.
It is noteworthy that the 3MST enzyme lacks extensive literature and until recently, no substance was described as its inhibitor. It is also well known that the use of molecules with inhibitory or amplifying activity in in vitro and in vivo experiments may reveal interesting results regarding the role of an enzyme in the physiology and pathophysiology of the studied organism. Therefore, the discovery of molecules with inhibitory activity on this enzyme or even the discovery of hit compounds which could be improved in order for stronger inhibitors to be made, would be of great significance. Given this, we tested the effect of different molecules characterized as tetracyclines on the enzyme activity. Our first results can be described as encouraging, as specific tetracyclines show inhibition of the 3MST. Further experiments allowed us to speculate the mechanism of these inhibitor molecules.
Keywords:
hydrogen sulfide, gasotransmitter,3-mercaptopyruvate sulfurtransferase, inhibitors, enzyme kinetics, tetracyclines, methylene blue assay
