Arthrobacter D-xylose isomerase: protein-engineered subunit interfaces.
Mutants of Arthrobacter D-xylose isomerase were constructed in which one or two disulphide bridges or additional salt bridges were introduced at the A-A* subunit interfaces. These showed no change in enzyme activity or stability compared with the wild-type enzyme. However, a Tyr253 mutant in which a disulphide bridge was introduced at the A-B* subunit interface showed reduced thermostability that was identical in both oxidized and reduced forms, and also reduced stability in urea. X-ray-crystallographic analysis of the Mn(2+)-xylitol form of oxidized Y253C (the Tyr253-->Cys mutant) showed a changed conformation of Glu185 and also alternative conformations for Asp254, which is a ligand to the Site-[2] metal ion. With fructose, Mg(2+)-Y253C has a similar Km to that of the wild-type, and its Vmax. is also similar below pH 6.4, but declined thereafter. In the presence of Co2+, Y253C has lower activity than wild-type at all pH values, but its activity also declines at alkaline pH. These results suggest that electrostatic repulsion from the new position of Glu185 causes Asp254 to move when His219 is unprotonated, thereby preventing M2+ binding at Site [2]. These results also suggest that subunit dissociation does not lie on the pathway of thermal inactivation of D-xylose isomerase, but that movements of active-site groups are a trigger for conformational changes that initiate the unfolding process.
Varsani
L
L
Centre for Biotechnology, Imperial College of Science, Technology and Medicine, London, U.K.
Cui
T
T
Rangarajan
M
M
Hartley
B S
BS
Goldberg
J
J
Collyer
C
C
Blow
D M
DM
D019747:Aldose-Ketose Isomerases; D001173:Arthrobacter / Q000201:enzymology*; D001483:Base Sequence; D001665:Binding Sites; D002238:Carbohydrate Epimerases / Q000737:chemistry* / Q000235:genetics; D003545:Cysteine; D004220:Disulfides / Q000378:metabolism; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011489:Protein Denaturation; D015202:Protein Engineering; D014443:Tyrosine; D014961:X-Ray Diffraction
D004220:Disulfides; D046911:Macromolecular Substances; D014443:Tyrosine; D002238:Carbohydrate Epimerases; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase; D003545:Cysteine