ARTHROBACTER D-XYLOSE ISOMERASE - CHEMICAL MODIFICATION OF CARBOXY GROUPS AND PROTEIN ENGINEERING OF PH OPTIMUM

To try to lower the pH optimum, the carboxy groups of Arthrobacter D-x ylose isomerase were coupled to glycinamide using a water-soluble carb odi-imide. In conditions that substituted all of the 59 carboxy groups in the denatured monomer, a maximum of 30 groups/monomer reacted in t he native enzyme, whether in presence or absence of ligands, and the e nzyme remained fully active and tetrameric throughout the coupling rea ction. Purification by fp.l.c. ion-exchange chromatography gave broad symmetrical peaks with increased pl, suggesting that the modified enzy mes are essentially homogeneous. However, they are less stable than na tive enzyme in 8 M urea or on heating ('melting points' of 59-degrees versus 73-degrees-C for the apoenzymes and 67-degrees versus 81.5-degr ees-C for the Mg2+-enzymes). Kinetic studies of the D-fructose isomera se activity at 30-degrees-C showed that the glycin-amidylated enzyme h ad unaltered activation constant for Mg2+, and K(m) was also similar t o that of the native enzyme at pH 7.3, but increased rapidly at higher pH rather than remaining constant. V(max) was constant from pH 6.2 to 8.0, suggesting a reduced pK(a) for His-219, which controls V(max) in the native enzyme (normally 6.0). Three mutants were constructed by p rotein engineering with a view to reducing the pH optimum of enzyme ac tivity. Two of these, Glu140 --> Lys and Asp189 --> Lys, could be dete cted in crude extracts of Escherichia coli by SDS/PAGE, but could not be purified, whereas mutant Trp136 --> Glu was produced as a tetramer in amounts similar to the wild-type enzyme. However, it did not show a ny enzyme activity and was less stable in 0-9 M urea gradient PAGE.