Investigation of Bi-Substrate Enzyme Kinetics for the Introductory Biochemistry Lab

Student: Krystal Scinto* and Khristopher White
Mentor: Benjamin Alper
Major: Chemistry 

Glutathione S-Transferase (GST) is a bi-substrate enzyme that plays an important role in drug detoxification. The enzyme’s activity is measured by colorimetric methods using 1-chloro-2,4-dinitrobenzene (CDNB), which is well suited to quantitative monitoring in real time using visible absorbance spectrometry. The conditions of this assay as previously employed in the Introductory Biochemistry Lab at Sacred Heart University were poorly suited to large scale kinetic analysis. Here, we are developing a tractable and highly reproducible large scale kinetic assay that is based on the CDNB method. We are also optimizing the conditions of this assay so that it can be applied within the educational environment and we are determining the optimal conditions for kinetic analysis to investigate the kinetic mechanism of GST. As a transferase, GST adopts an enzymatic mechanism that is thought to require at least two substrates:  glutathione, and the target compound to which it is transferred (various drugs, or other metabolites).  Over the course of our project, we will seek to investigate the enzymatic mechanism by which GST transfers glutathione onto its conjugation target, which in our research is CDNB.

*Honors Senior