Kinetic Studies of 2‐(2′‐hydroxyphenyl)benzenesulfinate desulfinase substrate analogs

Abstract

2‐(2′‐hydroxyphenyl)benzenesulfinate desulfinase (DszB) is an enzyme that catalyzes the cleavage of the carbon‐sulfur bond as the final step of the desulfurization of 2‐(2′‐hydroxyphenyl)benzenesulfinate (HPBS) producing sulfite and 2‐hydroxybiphenyl (HBP). This reaction is used to study the biodesulfurization of dibenzothiophene, the major organosulfur compound found in fossil fuels/petroleum. Previous studies in the Watkins lab suggested that halogenated analogs of HPBS are competitive inhibitors of DszB, so a coupled assay was used to test their effect on kinetic activity. The relative activity of these analogs was tested measure both products of the reaction. The rate of HBP formation was monitored using a fluorometric assay with an excitation at 288 nm and measuring emission at 414 nm. The rate of sulfite product formation was measured in the coupled assay with Sulfite Oxidase (SOX) by analyzing a change in absorbance at 540 nm. When testing the analogs HPBS‐Cl and HPBS‐Br for activity, no product was detected. When measuring enzyme activity in the presence of the halogenated analogs and the substrate HPBS, an increase in DszB activity was observed, with HPBS‐Cl and HPBS‐Br showing an increase product formation of 475% and 213%, respectively. Individually these HPBS analogs did not act as catalytic substrates or competitive inhibitors, however when coupled with HPBS the analogs acted as activators. Based on these results, we are exploring the structure of DszB to further understand how HPBS analogs can affect the desulfinase kinetics.Support or Funding InformationNSF CHE‐1757874This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.