Purification and characterisation of the phosphoglycerate kinase isoenzymes of Trypanosoma brucei expressed in Escherichia coli.
The Trypanosoma brucei phosphoglycerate kinase (PGK) glycosomal and cytosolic isoenzymes have been overexpressed in Escherichia coli and purified to near-homogeneity. Both enzymes were similar to the corresponding natural proteins with respect to their physicochemical and kinetic properties. In addition, a mutant of the glycosomal PGK lacking the 20 amino acid long C-terminal extension was overexpressed and purified. Various properties of this truncated glycosomal PGK were examined and it was found that in some aspects the protein behaved quite differently when compared with its natural counterpart. This was notably the case for the apparent Km for 3-phosphoglyceric acid, its sensitivity to inhibitors and its response to salts and guanidine HCl. However, its Vmax was found to be similar to that of the natural glycosomal PGK. These results suggest that the changes in the C-terminus caused a conformational change effecting the 3-phosphoglyceric acid binding site located at the N-terminal domain of the protein.
Zomer
A W
AW
Research Unit for Tropical Diseases, Christian de Duve Institute of Cellular Pathology and Laboratory of Biochemistry, Catholic University of Louvain, Brussels, Belgium.
Allert
S
S
Chevalier
N
N
Callens
M
M
Opperdoes
F R
FR
Michels
P A
PA
D000818:Animals; D003600:Cytosol / Q000201:enzymology; D004926:Escherichia coli / Q000235:genetics; D005988:Glyceric Acids / Q000378:metabolism; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D015388:Organelles / Q000201:enzymology; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D010735:Phosphoglycerate Kinase / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification*; D013498:Suramin / Q000494:pharmacology; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics
D005988:Glyceric Acids; D007527:Isoenzymes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D013498:Suramin; C005156:3-phosphoglycerate; D010735:Phosphoglycerate Kinase