Molecular cloning and analysis of two tandemly linked genes for pyruvate kinase of Trypanosoma brucei.
In Trypanosoma brucei (stock 427) genes encoding the glycolytic enzyme pyruvate kinase are present on two homologous chromosomes. We have cloned and characterized one of the alleles. Two large, tandemly arranged open reading frames were found, each coding for a pyruvate kinase polypeptide of 498 amino acids. The gene sequences differ at 15 positions, resulting in five amino acid substitutions. The calculated molecular masses of the polypeptides are 54,378 Da and 54,363 Da. These values are somewhat smaller than those reported for the subunit molecular mass of the purified protein, which is 57-59 kDa. However, in vitro translation of the DNA region corresponding to the open reading frame, and translation of the RNA in a wheat-germ lysate, yielded a product that comigrated exactly with the native polypeptide in SDS/PAGE. The overall identity between the sequences of the trypanosomal enzyme and the enzymes from other sources is 41-51%. The conserved residues are not equally distributed over the polypeptide. The primary structure of domains A and, to a lesser extent, B, which constitute the active site, are rather well conserved. In contrast, the sequence of domain C, which supposedly is involved in the regulation of the enzyme activity, is much more variable. The cytosolically located pyruvate kinase of T. brucei lacks the specific features found in the majority of the glycolytic enzymes of this organism that are sequestered in a microbody-like organelle, the glycosome. It has neither a relatively high subunit molecular mass, due to unique insertions or terminal extensions, nor a high excess of positively charged amino acids. The polypeptide is shorter than that of most other pyruvate kinases and the calculated net charge is only +3.
Allert
S
S
International Institute of Cellular and Molecular Pathology, Research Unit for Tropical Diseases, Brussels, Belgium.
Ernest
I
I
Poliszczak
A
A
Opperdoes
F R
FR
Michels
P A
PA
D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D016054:DNA, Protozoan / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011770:Pyruvate Kinase / Q000235:genetics*; D012091:Repetitive Sequences, Nucleic Acid; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics
D016054:DNA, Protozoan; D011770:Pyruvate Kinase