ROLE OF ELECTROSTATICS AT THE CATALYTIC METAL-BINDING SITE IN XYLOSE ISOMERASE ACTION - CA2-INHIBITION AND METAL COMPETENCE IN THE DOUBLE MUTANT D254E()D256E/

The catalytic metal binding site of xylose isomerase from Arthrobacter B3728 was modified by protein engineering to diminish the inhibitory effect of Ca2+ and to study the competence of metals on catalysis. To exclude Ca2+ from Site 2 a double mutant D254E/D256E was designed with reduced space available for binding. In order to elucidate structural consequences of the mutation the binary complex of the mutant with Mg 2+ as well as ternary complexes with bivalent metal ions and the open- chain inhibitor xylitol were crystallized for x-ray studies. We determ ined the crystal structures of the ternary complexes containing Mg2+, Mn2+, and Ca2+ at 2.2 to 2.5 Angstrom resolutions, and refined them to R factors of 16.3, 16.6, and 19.1, respectively. We found that all me tals are liganded by both engineered glutamates as well as by atoms O1 and O2 of the inhibitor. The similarity of the coordination of Ca2+ t o that of the cofactors as well as results with Be2+ weaken the assump tion that geometry differences should account for the catalytic noncom petence of this ion. Kinetic results of the D254E/D256E mutant enzyme showed that the significant decrease in Ca2+ inhibition was accompanie d by a similar reduction in the enzymatic activity. Qualitative argume ntation, based on the protein electrostatic potential, indicates that the proximity of the negative side chains to the substrate significant ly reduces the electrostatic stabilization of the transition state. Fu rthermore, due to the smaller size of the catalytic metal site, no wat er molecule, coordinating the metal, could be observed in ternary comp lexes of the double mutant. Consequently, the proton shuttle step in t he overall mechanism should differ from that in the wild type. These e ffects can account for the observed decrease in catalytic efficiency o f the D254E/D256E mutant enzyme. (C) 1997 Wiley-Liss, Inc.