ARTHROBACTER D-XYLOSE ISOMERASE - PROTEIN-ENGINEERED SUBUNIT INTERFACES

Mutants of Arthrobacter D-xylose isomerase were constructed in which o ne or two disulphide bridges or additional salt bridges were introduce d at the A-A subunit interfaces. These showed no change in enzyme act ivity or stability compared with the wild-type enzyme. However, a Tyr( 253) mutant in which a disulphide bridge was introduced at the A-B su bunit interface showed reduced thermostability that was identical in b oth oxidized and reduced forms, and also reduced stability in urea. X- ray-crystallo-graphic analysis of the Mn2+-xylitol form of oxidized Y2 53C (the Tyr253 --> Cys mutant) showed a changed conformation of Glu18 5 and also alternative conformations for Asp254, Which is a ligand to the Site-[2] metal ion. With fructose, Mg2+-Y253C has a similar K(m) t o that of the wild-type, and its V(max) is also similar below pH 6.4, but declined thereafter. In the presence of Co2+, Y253C has lower acti vity than wild-type at all pH values, but its activity also declines a t alkaline pH. These results suggest that electrostatic repulsion from the new position of Glu185 causes Asp254 to move when His219 is unpro tonated, thereby preventing M2+ binding at Site [2]. These results als o suggest that subunit dissociation does not lie on the pathway of the rmal inactivation Of D-xylose isomerase, but that movements of active- site groups are a trigger for conformational changes that initiate the unfolding process.