Molecular and biochemical characterization of novel glucokinases from Trypanosoma cruzi and Leishmania spp.
Glucokinase genes, found in the genome databases of Trypanosoma cruzi and Leishmania major, were cloned and sequenced. Their expression in Escherichia coli resulted in the synthesis of soluble and active enzymes, TcGlcK and LmjGlcK, with a molecular mass of 43 kDa and 46 kDa, respectively. The enzymes were purified, and values of their kinetic parameters determined. The K(m) values for glucose were 1.0 mM for TcGlcK and 3.3 mM for LmjGlcK. For ATP, the K(m) values were 0.36 mM (TcGlcK) and 0.35 mM (LmjGlcK). A lower K(m) value for glucose (2.55 mM) was found when the (His)(6)-tag was removed from the recombinant LmjGlcK, whereas the TcGlcK retained the same value. The V(max)'s of the T. cruzi and L. major GlcKs were 36.3 and 30.9 U/mg of protein, respectively. No inhibition was exerted by glucose-6-phosphate. Similarly, no inhibition by inorganic pyrophosphate was found in contrast to previous observations made for the T. cruzi and L. mexicana hexokinases. Both trypanosomatid enzymes were only able to phosphorylate glucose indicating that they are true glucokinases. Gel-filtration chromatography showed that the GlcK of both trypanosomatids may occur as a monomer or dimer, dependent on the protein concentration. Both GlcK sequences have a type-1 peroxisome-targeting signal. Indeed, they were shown to be present inside glycosomes using three different methods. These glucokinases present highest, albeit still a moderate 24% sequence identity with their counterpart from Trichomonas vaginalis, which has been classified into group A of the hexokinase family. This group comprises mainly eubacterial and cyanobacterial glucokinases. Indeed, multiple sequence comparisons, as well as kinetic properties, strongly support the notion that these trypanosomatid enzymes belong to group A of the hexokinases, in which they, according to a phylogenetic analysis, form a separate cluster.
Cáceres
Ana Judith
AJ
Unidad de Bioquímica de Parásitos, Centro de Ingeniería Genética, Facultad de Ciencias, Universidad de Los Andes, Mérida 5101, Venezuela.
Quiñones
Wilfredo
W
Gualdrón
Melisa
M
Cordeiro
Artur
A
Avilán
Luisana
L
Michels
Paul A M
PA
Concepción
Juan Luis
JL
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D019281:Dimerization; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D005941:Glucokinase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D019298:Glucose-6-Phosphate / Q000494:pharmacology; D007700:Kinetics; D018320:Leishmania major / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D020675:Peroxisomes / Q000737:chemistry; D010710:Phosphates / Q000494:pharmacology; D010802:Phylogeny; D021382:Protein Sorting Signals / Q000235:genetics; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014246:Trichomonas vaginalis / Q000235:genetics; D014349:Trypanosoma cruzi / Q000201:enzymology* / Q000235:genetics
D004791:Enzyme Inhibitors; D010710:Phosphates; D021382:Protein Sorting Signals; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D019298:Glucose-6-Phosphate; D000255:Adenosine Triphosphate; D005941:Glucokinase; D005947:Glucose