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 pyr uvate kinase in Escherichia coli. The enzyme was purified to near-homo geneity from the bacterial lysate by first removing nucleic acids and contaminating proteins by protamine sulfate precipitation and subseque nt passage over a phosphocellulose column. The purified protein is ess entially indistinguishable in its physicochemical and kinetic properti es from the enzyme purified from trypanosomes. Furthermore, experiment s were undertaken to locate the binding site of the allosteric effecto r fructose 2,6-bisphosphate. Regulation of pyruvate kinase by this eff ector is unique to trypanosomes and related protozoan organisms. There fore, 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 prote ins were produced and purified, using the same methodology as for the wildtype pyruvate kinase. Some mutants showed only minor changes in th e activation by the effector. However, substitution of Arg22 by Gly re sulted in a 9.2-fold higher S-0.5 for phosphoenolpyruvate and a signif icantly smaller k(cat) than the wild-type enzyme. Furthermore, the app arent affinity of this mutant for the allosteric effecters fructose 1, 6-bisphosphate and fructose 2,6-bisphosphate was 8.2- and 5.2-fold low er than that of its wild-type counterpart. Effector binding was also a ffected, although to a lesser extent, in a mutant Phe463Val. These dat a indicate that particularly residue Arg22, but also Phe463, are someh ow involved in the binding of the allosteric effecters. (C) 1998 Acade mic Press.