Determining the effect of dithiolethione compounds on the activity of human glyceraldehyde‐3‐phosphate dehydrogenase

Abstract

Dithiolethione compounds have chemopreventive, cytoprotective, and antimitogenic effects. We have shown that some of these compounds interact with glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH). In cells, GAPDH is an abundant and multifaceted protein involved in glycolysis, cell death, DNA repair, regulation of gene expression, tRNA export, membrane fusion and transport. As a result of its plethora of roles, there is an incentive in understanding how dithiolethiones affect GAPDH activity and function. Dithiolethiones can directly or indirectly modify cysteine residues in protein targets. GAPDH contains three cysteine residues that are susceptible to thiol‐modifying agents. Therefore, we set out to examine how the dithiolethione, ADT‐OH, affects GAPDH activity.Human GAPDH was overexpressed in bacterial cells and purified by ion‐exchange and affinity chromatography. We measured the catalytic activity of GAPDH in the absence and the presence of ADT‐OH, and in the absence of DTT. Comparison of the kinetics parameters for unmodified GAPDH and GAPDH modified with increasing concentrations of ADT‐OH reveals a concentration‐dependent decrease of GAPDH catalytic activity.Our next step will be to determine the exact mechanism of ADT‐OH modification of GAPDH by combining site‐directed mutagenesis, mass‐spectrometry, circular dichroism, and x‐ray crystallography.FundingUMBC DRIF SRAIS (EG)This research was supported in part by a grant to UMBC from the Howard Hughes Medical Institute through the Precollege and Undergraduate Science Education Program.