Characterization of a divergent glycosomal microbody phosphoglycerate kinase from Trypanosoma brucei.
There are 3 loci in the phosphoglycerate kinase (PGK) gene complex of Trypanosoma brucei. The PGK-A gene product, which we term 56PGK, is targeted to glycosomal microbodies and is highly homologous to the parasite's 2 known PGKs (one cytoplasmic and one glycosomal). However, 56PGK contains an 80 amino acid insertion as well as numerous substitutions compared to the other PGKs. The complementation and kinetic analyses described here demonstrate that 56PGK is an authentic phosphoglycerate kinase--the largest yet described. When expressed in Escherichia coli, 56PGK complements the pgk- phenotype. 56PGK was expressed as a fusion protein and purified to near homogeneity. The Michaelis constants are similar to those of other PGKs, being 0.12 and 2.4 mM for Mg-ATP and 3-phosphoglycerate, respectively. As with other T. brucei PGKs, ATP but not GTP or ITP can serve as a phosphate donor during catalysis. No evidence was obtained for phosphate transfer to atypical substrates. 56PGK shows sulfate inhibition at all concentrations tested, rather than the sulfate activation observed with yeast PGK.
Alexander
K
K
Seattle Biomedical Research Institute, WA 98109-1651.
Parsons
M
M
D000595:Amino Acid Sequence; D000818:Animals; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D017125:Genes, Protozoan; D005816:Genetic Complementation Test; D007700:Kinetics; D008830:Microbodies / Q000201:enzymology; D008969:Molecular Sequence Data; D010641:Phenotype; D010735:Phosphoglycerate Kinase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics*
D011993:Recombinant Fusion Proteins; D010735:Phosphoglycerate Kinase