title: Biochemical characterization of highly active Trypanosoma brucei gambiense glycerol kinase, a promising drug target.
abstract: Human African trypanosomes are blood parasites that cause sleeping sickness, a debilitating disease in sub-Saharan Africa. Glycerol kinase (GK) of these parasites additionally possesses a novel property of reverse catalysis. GK is essential to blood stream form trypanosome, and therefore a promising drug target. Here, utilizing recombinant DNA technology an optimized procedure for obtaining large amount of the purified protein was established. Furthermore, biochemical data on its enzymology are reported. The protein was maximally active at pH 6.8 over a temperature range of 25-70°C, with activation energy of 34.02 ± 0.31 kJ mol(-1). The enzyme catalyses a reversible bisubstrate [ADP and glycerol 3-phosphate (G3P)]-biproduct (ATP and glycerol) reaction. It has Km of 0.90 and 5.54 mM for ADP and G3P, respectively, and Vmax of 25.3 and 20.0 µmol min(-1) mg(-1), respectively. Unexpectedly, the enzyme lost more than 50% of its activity in 48 h at 4°C in 0.1 M sodium phosphate buffer pH 6.8 containing 10 mM MgSO4. However, perfect stabilization of the GK for more than 4 weeks was achieved in the presence of its natural ligands and cofactor. Using this stabilized protein, crystals of trypanosome GK with better resolution were obtained. This will accelerate the success of GK inhibitor development for drug design.
authors.affiliation: Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Emmanuel Oluwadare
authors.initials: EO
authors.lastname: Balogun
authors.forename: Daniel Ken
authors.initials: DK
authors.lastname: Inaoka
authors.forename: Tomoo
authors.initials: T
authors.lastname: Shiba
authors.forename: Yasutoshi
authors.initials: Y
authors.lastname: Kido
authors.forename: Takeshi
authors.initials: T
authors.lastname: Nara
authors.forename: Takashi
authors.initials: T
authors.lastname: Aoki
authors.forename: Teruki
authors.initials: T
authors.lastname: Honma
authors.forename: Akiko
authors.initials: A
authors.lastname: Tanaka
authors.forename: Masayuki
authors.initials: M
authors.lastname: Inoue
authors.forename: Shigeru
authors.initials: S
authors.lastname: Matsuoka
authors.forename: Paul A M
authors.initials: PA
authors.lastname: Michels
authors.forename: Shigeharu
authors.initials: S
authors.lastname: Harada
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Kita
mesh_terms: D003460:Crystallization; D018360:Crystallography, X-Ray; D015195:Drug Design; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D017125:Genes, Protozoan; D005991:Glycerol Kinase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D015800:Protozoan Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014347:Trypanosoma brucei gambiense / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity; D014353:Trypanosomiasis, African / Q000188:drug therapy / Q000201:enzymology / Q000469:parasitology
chemical_list: D004791:Enzyme Inhibitors; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D005991:Glycerol Kinase
keywords: African trypanosomes; characterization; crystallization; drug design; glycerol kinase