Molecular and biochemical characterization of hexokinase from Trypanosoma cruzi.
The Trypanosoma cruzi hexokinase gene has been cloned, sequenced, and expressed as an active enzyme in Escherichia coli. Sequence analysis revealed 67% identity with its counterpart in Trypanosoma brucei but low similarity with all other available hexokinase sequences including those of human. It contains an N-terminal peroxisome-targeting signal (PTS-2) and has a calculated basic isoelectric point (pI = 9.67), a feature often associated with glycosomal proteins. The polypeptide has a predicted mass of approximately 50 kDa similar to that of many non-vertebrate hexokinases and the vertebrate hexokinase isoenzyme IV. The natural enzyme was purified to homogeneity from T. cruzi epimastigotes and appeared to exist in several aggregation states, an apparent tetramer being the predominant form. Its kinetic properties were compared with those of the purified recombinant protein. Higher K(m) values for glucose and ATP were found for the (His)(6)-tag-containing recombinant hexokinase. However, removal of the tag produced an enzyme displaying similar values as the natural enzyme (K(m) for glucose = 43 and 60 microM for the natural and the recombinant protein, respectively). None of these enzymes presented activity with fructose. As reported previously for hexokinases from several trypanosomatids, no inhibition was exerted by glucose 6-phosphate (G6-P). In contrast, a mixed-type inhibition was observed with inorganic pyrophosphate (PPi, K(i) = 0.5mM).
Cáceres
Ana Judith
AJ
Unidad de Bioquímica de Parásitos, Centro de Ingeniería Genética, Facultad de Ciencias, Universidad de Los Andes, Mérida 5101, Venezuela.
Portillo
Ramon
R
Acosta
Hector
H
Rosales
David
D
Quiñones
Wilfredo
W
Avilan
Luisana
L
Salazar
Leiria
L
Dubourdieu
Michel
M
Michels
Paul A M
PA
Concepción
Juan Luis
JL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006593:Hexokinase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014349:Trypanosoma cruzi / Q000201:enzymology* / Q000235:genetics
D017931:DNA Primers; D007527:Isoenzymes; D011994:Recombinant Proteins; D006593:Hexokinase