title: Effect of lysine to alanine mutations on the phosphate activation and BPTES inhibition of glutaminase.
abstract: The GLS1 gene encodes a mitochondrial glutaminase that is highly expressed in brain, kidney, small intestine and many transformed cells. Recent studies have identified multiple lysine residues in glutaminase that are sites of N-acetylation. Interestingly, these sites are located within either a loop segment that regulates access of glutamine to the active site or the dimer:dimer interface that participates in the phosphate-dependent oligomerization and activation of the enzyme. These two segments also contain the binding sites for bis-2[5-phenylacetamido-1,2,4-thiadiazol-2-yl]ethylsulfide (BPTES), a highly specific and potent uncompetitive inhibitor of this glutaminase. BPTES is also the lead compound for development of novel cancer chemotherapeutic agents. To provide a preliminary assessment of the potential effects of N-acetylation, the corresponding lysine to alanine mutations were constructed in the hGACΔ1 plasmid. The wild type and mutated proteins were purified by Ni(+)-affinity chromatography and their phosphate activation and BPTES inhibition profiles were analyzed. Two of the alanine substitutions in the loop segment (K311A and K328A) and the one in the dimer:dimer interface (K396A) form enzymes that require greater concentrations of phosphate to produce half-maximal activation and exhibit greater sensitivity to BPTES inhibition. By contrast, the K320A mutation results in a glutaminase that exhibits near maximal activity in the absence of phosphate and is not inhibited by BPTES. Thus, lysine N-acetylation may contribute to the acute regulation of glutaminase activity in various tissues and alter the efficacy of BPTES-type inhibitors.
authors.affiliation: Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523-1870, United States.
authors.forename: Charles J
authors.initials: CJ
authors.lastname: McDonald
authors.affiliation: Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523-1870, United States.
authors.forename: Eric
authors.initials: E
authors.lastname: Acheff
authors.affiliation: Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523-1870, United States.
authors.forename: Ryan
authors.initials: R
authors.lastname: Kennedy
authors.affiliation: Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523-1870, United States.
authors.forename: Lynn
authors.initials: L
authors.lastname: Taylor
authors.affiliation: Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523-1870, United States. Electronic address: Norman.Curthoys@Colostate.edu.
authors.forename: Norman P
authors.initials: NP
authors.lastname: Curthoys
mesh_terms: D000409:Alanine / Q000235:genetics*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D005972:Glutaminase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008239:Lysine / Q000235:genetics*; D009154:Mutation / Q000235:genetics*; D010710:Phosphates / Q000494:pharmacology*; D013440:Sulfides / Q000494:pharmacology*; D013830:Thiadiazoles / Q000494:pharmacology*
chemical_list: D010710:Phosphates; D013440:Sulfides; D013830:Thiadiazoles; C523193:bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide; D005972:Glutaminase; D008239:Lysine; D000409:Alanine
keywords: Alanine substitution; BPTES inhibition; Glutaminase; Lysine N-acetylation; Ni(+)-affinity chromatography; Phosphate activation