SELENOMETHIONINE LABELING OF PHOSPHOMANNOSE ISOMERASE CHANGES ITS KINETIC-PROPERTIES

Phosphomannose isomerase (PMI) is an essential enzyme in the early ste ps of the protein glycosylation pathway in both prokaryotes and eukary otes. Lack of the enzyme is lethal for fungal organisms and it is thus a potential fungicidal target. To facilitate the solution of the thre e-dimensional structure of the enzyme from the pathogen Candida albica ns, we have produced the recombinant: selenomethionine-labelled enzyme (SeMet-PMI). DL41, a methionine auxotroph Escherichia coli strain, wa s transformed with a PMI expression plasmid and grown on an enriched s elenomethionine-containing medium to high-cell densities. The SeMet-PM I protein has been purified and found by amino acid analysis to have i ts methionine residues replaced by selenomethionine residues. Electros pray mass spectroscopy showed a major species of 49 063 +/- 10 Da for SeMet-PMI compared to 48735 +/- 6 Da for the normal recombinant enzyme , accounting for the incorporation of seven selenomethionine residues. SeMet-PMI crystallised isomorphously with the normal PMI protein and the crystals diffract to 0.23 nm. Kinetic characterisation of SeMet-PM I showed that its K-m for the substrate mannose-6-phosphate was fourfo ld higher than that of its methionine-containing counterpart. The inhi bition constant for zinc ions was also increased by a similar factor. However, the V-max was unaltered. These results suggested that one or more methionine residues must be in close proximity to the substrate-b inding pocket in the active site, rendering substrate access more diff icult compared to the normal enzyme. This hypothesis was confirmed by the finding of four methionine residues lying along one wall of the ac tive site.