title: Assembly of heterohexameric trypanosome hexokinases reveals that hexokinase 2 is a regulable enzyme.
abstract: Glycolysis is essential to Trypanosoma brucei, the protozoan parasite that causes African sleeping sickness in humans and nagana in cattle. Hexokinase (HK), the first enzyme in glycolysis, catalyzes the phosphorylation of glucose to form glucose 6-phosphate. T. brucei harbors two HKs that are 98% identical at the amino acid level, T. brucei hexokinase 1 (TbHK1) and TbHK2. Recombinant TbHK1 (rTbHK1) has HK activity, whereas rTbHK2 does not. Unlike other eukaryotic HKs, TbHK1 is not subject to inhibition by ADP and glucose 6-phosphate. However, TbHK1 is inhibited by myristate, a critical fatty acid in T. brucei biology. We report here that rTbHKs, similar to authentic TbHK, form oligomers. Myristate dissociated these assemblies when incubated with either ATP or glucose. Furthermore, oligomer disruption was reversible by removal of myristate. Mixing of rTbHK1 and rTbHK2 monomers followed by reassembly yielded enzyme with an approximately 3-fold increase in specific activity compared with similarly treated rTbHK1 alone. Surprisingly, reassembly of rTbHK2 with an inactive rTbHK1 variant yielded an active HK, revealing for the first time that rTbHK2 is competent for HK activity. Finally, pyrophosphate inhibits active reassembled rTbHK2 oligomers but not oligomeric rTbHK1, suggesting that the two enzymes have distinct regulatory mechanisms.
authors.affiliation: Department of Genetics and Biochemistry, Clemson University, 51 New Cherry Street, Clemson, SC 29634, USA.
authors.forename: Jeremy W
authors.initials: JW
authors.lastname: Chambers
authors.forename: Margaret T
authors.initials: MT
authors.lastname: Kearns
authors.forename: Meredith T
authors.initials: MT
authors.lastname: Morris
authors.forename: James C
authors.initials: JC
authors.lastname: Morris
mesh_terms: D000818:Animals; D004789:Enzyme Activation / Q000502:physiology; D005947:Glucose / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019298:Glucose-6-Phosphate / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006019:Glycolysis / Q000502:physiology; D006593:Hexokinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019814:Myristic Acid / Q000737:chemistry* / Q000378:metabolism; D020836:Protein Structure, Quaternary / Q000502:physiology; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics; D014353:Trypanosomiasis, African / Q000201:enzymology / Q000235:genetics
chemical_list: D015800:Protozoan Proteins; D011994:Recombinant Proteins; D019814:Myristic Acid; D019298:Glucose-6-Phosphate; D006593:Hexokinase; D005947:Glucose