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-xylose isomerase were coupled to glycinamide using a water-soluble carbodi-imide. In conditions that substituted all of the 59 carboxy groups in the denatured monomer, a maximum of 30 groups/monomer reacted in the native enzyme, whether in presence or absence of ligands, and the enzyme remained fully active and tetrameric throughout the coupling reaction. Purification by f.p.l.c. ion-exchange chromatography gave broad symmetrical peaks with increased pI, suggesting that the modified enzymes are essentially homogeneous. However, they are less stable than native 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 degrees C for the Mg(2+)-enzymes). Kinetic studies of the D-fructose isomerase activity at 30 degrees C showed that the glycinamidylated enzyme had unaltered activation constant for Mg2+, and Km was also similar to that of the native enzyme at pH 7.3, but increased rapidly at higher pH rather than remaining constant. Vmax. was constant from pH 6.2 to 8.0, suggesting a reduced pKa for His-219, which controls Vmax. in the native enzyme (normally 6.0). Three mutants were constructed by protein engineering with a view to reducing the pH optimum of enzyme activity. Two of these, Glu140-->Lys and Asp189-->Lys, could be detected 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 any enzyme activity and was less stable in 0-9 M urea gradient PAGE.
Siddiqui
K S
KS
Centre for Biotechnology, Imperial College of Science, Technology and Medicine, London, U.K.
Loviny-Anderton
T
T
Rangarajan
M
M
Hartley
B S
BS
D019747:Aldose-Ketose Isomerases; D000577:Amides / Q000737:chemistry; D001173:Arthrobacter / Q000201:enzymology*; D001483:Base Sequence; D002238:Carbohydrate Epimerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005971:Glutamates / Q000737:chemistry; D018698:Glutamic Acid; D005998:Glycine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D015202:Protein Engineering; D013820:Thermolysin; D014364:Tryptophan / Q000737:chemistry
D000577:Amides; D005971:Glutamates; D009838:Oligodeoxyribonucleotides; D018698:Glutamic Acid; D014364:Tryptophan; D013820:Thermolysin; D002238:Carbohydrate Epimerases; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase; D005998:Glycine