Pyruvate kinase of Trypanosoma brucei: overexpression, purification, and functional characterization of wild-type and mutated enzyme.
A procedure was developed for overexpression of Trypanosoma brucei pyruvate kinase in Escherichia coli. The enzyme was purified to near-homogeneity from the bacterial lysate by first removing nucleic acids and contaminating proteins by protamine sulfate precipitation and subsequent passage over a phosphocellulose column. The purified protein is essentially indistinguishable in its physicochemical and kinetic properties from the enzyme purified from trypanosomes. Furthermore, experiments were undertaken to locate the binding site of the allosteric effector fructose 2,6-bisphosphate. Regulation of pyruvate kinase by this effector is unique to trypanosomes and related protozoan organisms. Therefore, a three-dimensional structure model of the enzyme was made, and a putative effector-binding site could be identified in an interdomain cleft. Four residues in this cleft were mutated, and the mutant proteins were produced and purified, using the same methodology as for the wild-type pyruvate kinase. Some mutants showed only minor changes in the activation by the effector. However, substitution of Arg22 by Gly resulted in a 9.2-fold higher S(0.5) for phosphoenolpyruvate and a significantly smaller kcat than the wild-type enzyme. Furthermore, the apparent affinity of this mutant for the allosteric effectors fructose 1,6-bisphosphate and fructose 2,6-bisphosphate was 8.2- and 5.2-fold lower than that of its wild-type counterpart. Effector binding was also affected, although to a lesser extent, in a mutant Phe463Val. These data indicate that particularly residue Arg22, but also Phe463, are somehow involved in the binding of the allosteric effectors.
Research Unit for Tropical Diseases, Christian de Duve Institute of Cellular Pathology and Laboratory of Biochemistry, Brussels, Belgium.
I
I
Ernest
M
M
Callens
A D
AD
Uttaro
N
N
Chevalier
F R
FR
Opperdoes
H
H
Muirhead
P A
PA
Michels
D000818:Animals; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D007525:Isoelectric Focusing; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011489:Protein Denaturation; D011770:Pyruvate Kinase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014346:Trypanosoma brucei brucei / Q000201:enzymology*
D011994:Recombinant Proteins; D011770:Pyruvate Kinase