Defining the active site of 2-(2'-hydroxyphenyl) benzenesulfinate desulfinase
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Diminishing resources of light crude oils containing low sulfur fractions have compelled petroleum refineries to process sour crude oils. Current sulfur removal processes include the use of hydrodesulfurization. Hydrodesulfurization uses an inorganic catalyst, hydrogen, high temperature and pressure to remove sulfur from petroleum leaving the sulfur in the form of hydrogen sulfide, resulting in environmental problems. Since larger volumes of sour crude oils, in the form of middle distillates, are being used in processing, the use of hydrodesulfurization has become costly and inefficient. Additionally, governmental regulation of the allowable sulfur content in refined fossil fuels has become more stringent. These trends have led scientists to research new desulfurization methods.
One such method is biodesulfurization, which utilizes microbes that possess carbon-sulfur bond cleavage capabilities to remove the sulfur. These microbes utilize multiple enzyme pathways to perform the desulfurization of compounds. An example of a carbon-sulfur bond cleaving organism is Rhodococcus erythropolis IGTS8. R. erythropolis uses a metabolic pathway consisting of four enzymes. This pathway catalyzes the desulfurization of dibenzothiophene to sulfite and hydroxybiphenol. The research conducted for this project has focused on the enzyme found in the final step of the pathway. The enzyme, known as 2-(2' -hydroxyphenyl) benzenesulfinate desulfinase (HPBS desulfinase), is considered the rate limiting step in the pathway. The initial phase of this study included the purification ofHPBS desulfinase. The enzyme was purified 260-fold from R. erythropolis. Purification of the enzyme was monitored using UV -Vis, spectrofluorimetric assays, and SDS-PAGE. In order to define the active site of HPBS desulfinase, it was necessary to determine what types of compounds could be used as substrates or inhibitors. Twenty-one commercially available analogs of both the substrate (HPBS) and the product (HBP) were tested, along with two synthesized substrate analogs, and none were found to act as substrates. However, several behaved as competent inhibitors, and the two synthesized analogs increased the activity of the enzyme. Analysis of the two synthesized analogs was performed using 13C NMR, 1H NMR, mass spectroscopy, TLC, and pKa determination. Further studies were conducted using thiourea dioxide and CuCh, which had both previously been shown to inhibit HPBS desulfinase. The results from the current study agree. In addition, it was determined that CuCh binds to the cysteine in the active site and not the sulfonic acid on the substrate. Chemical modification of the cysteine in the active site using DTNB showed that once modified, the enzyme showed no activity in the presence of substrate. Finally, a fluorescence method of determining inhibitor binding was developed and a Ko of 1.6 µM was calculated for 1, 8-naphthosultam' s ( an inhibitor) binding to the enzyme.
CitationChambers, R. L. (2003). Defining the active site of 2-(2'-hydroxyphenyl) benzenesulfinate desulfinase (Unpublished thesis). Southwest Texas State University, San Marcos, Texas.
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