Activity of a Second Trypanosoma brucei Hexokinase Is Controlled by an 18-Amino-Acid C-Terminal Tail

Abstract

Trypanosoma brucei expresses two hexokinases that are 98% identical, namely, TbHK1 and TbHK2. Homozygous null TbHK2؊/؊ procyclic-form parasites exhibit an increased doubling time, a change in cell morphology, and, surprisingly, a twofold increase in cellular hexokinase activity. Recombinant TbHK1 enzymatic activity is similar to that of other hexokinases, with apparent K m values for glucose and ATP of 0.09 ؎ 0.02 mM and 0.28 ؎ 0.1 mM, respectively. The k cat value for TbHK1 is 2.9 ؋ 10 4 min ؊1 . TbHK1 can use mannose, fructose, 2-deoxyglucose, and glucosamine as substrates. In addition, TbHK1 is inhibited by fatty acids, with lauric, myristic, and palmitic acids being the most potent (with 50% inhibitory concentrations of 75.8, 78.4, and 62.4 M, respectively). In contrast to TbHK1, recombinant TbHK2 lacks detectable enzymatic activity. Seven of the 10 amino acid differences between TbHK1 and TbHK2 lie within the C-terminal 18 amino acids of the polypeptides. Modeling of the proteins maps the C-terminal tails near the interdomain cleft of the enzyme that participates in the conformational change of the enzyme upon substrate binding. Replacing the last 18 amino acids of TbHK2 with the corresponding residues of TbHK1 yields an active recombinant protein with kinetic properties similar to those of TbHK1. Conversely, replacing the C-terminal tail of TbHK1 with the TbHK2 tail inactivates the enzyme. These findings suggest that the C-terminal tail of TbHK1 is important for hexokinase activity. The altered C-terminal tail of TbHK2, along with the phenotype of the knockout parasites, suggests a distinct function for the protein.

Enzymatic properties of the N- and C-terminal halves of human hexokinase II

“…The process is monitored at 340 nm. One unit of HK activity is defined as the amount of enzyme that catalyzes the production of 1 μmol of G6P or ADP per minute at 30 o C. Kinetic parameters were determined using LineweaverBurke and Eadie-Hofstee analyses, with Km determined using at least five experiments, as described previously (26,27).…”

Interrogating a Hexokinase-Selected Small-Molecule Library for Inhibitors of Plasmodium falciparum Hexokinase

“…The recombinant enzyme, which was purified to ϳ95% homogeneity (as determined by Coomassie staining of an SDS-PAGE gel), displayed Michaelis-Menten kinetics when substrate levels were increased ( Fig. 2A) (9). PfHK had an affinity for MgCl 2 (apparent K m ϭ 2.7 Ϯ 0.4 mM) similar to that of TbHK1 (apparent K m ϭ 0.92 Ϯ 0.21 mM).…”

“…HK assays were performed in triplicate as described elsewhere, using a coupled reaction to measure enzyme activity (9,10). In short, the coupled assay uses glucose-6-phosphate dehydrogenase (G6PDH) to convert glucose-6-phosphate (G6P) generated by HK to 6-phosphogluconate with coincident reduction of NADP to NADPH, which is monitored spectrophotometrically at 340 nm.…”

“…Recombinant PfHK, an ϳ55.3-kDa protein, was purified, following a protocol developed for heterologous expression and purifica-tion of a HK from the African trypanosome. Briefly, a 10-ml bacterial culture of Escherichia coli M15(pREP) harboring pQE30PfHK with PfHK cloned in frame of a 6-His sequence (9) was used to inoculate a 1-liter culture, which was grown to an optical density at 600 nm (OD 600 ) of ϳ1 and then induced for 24 h at room temperature (RT) with 500 M isopropyl ␤-D-1-thiogalactopyranoside (IPTG) and purified as described previously (9).…”

Assembly of Heterohexameric Trypanosome Hexokinases Reveals That Hexokinase 2 Is a Regulable Enzyme

“…Recombinant TbHK1 (rTbHK1) exhibits HK activity in vitro (7,8). Furthermore, RNA interference (RNAi) of TbHK1 leads to a reduction in HK activity in both PF and BSF parasites and is lethal to BSF parasites (4,9).…”