BPTES inhibition of hGA_124–551, a truncated form of human kidney-type glutaminase

Abstract

The initial transcript of the GLS1 gene undergoes alternative splicing to produce two glutaminase variants (KGA and GAC) that contain unique C-terminal sequences. A truncated form of human glutaminase (hGA_124–551) that lacks either C-terminal sequence was expressed in E.Coli and purified. This construct exhibits a hyperbolic glutamine saturation profile (K_m of 1.6 mM). BPTES, bis-2[5-phenylacetamido-1,2,4-thiadiazol-2-yl]ethylsulfide, functions as a potent uncompetitive inhibitor of this construct (K_i of 0.2 µM). The hGA_124–551 is inactive in the absence of phosphate, but exhibits a hyperbolic phosphate-dependent activation profile that is also inhibited by BPTES. Gel filtration studies indicate that hGA_124–551 forms a dimer in the absence or presence of 100 mM phosphate, whereas addition of BPTES causes the formation of an inactive tetramer. The combined data indicate that BPTES inhibits human glutaminase by a novel mechanism and that BPTES is a potential lead compound for development of an effective cancer chemotherapeutic agent.

Keywords::

• Recombinant glutaminase
• kinetic analysis
• uncompetitive inhibitor
• oligomerization
• cancer chemotherapy