Role of electrostatics at the catalytic metal binding site in xylose isomerase action: Ca2+-inhibition and metal competence in the double mutant D254E/D256E

Monika Fuxreiter, Zsolt Böcskei, Anikó Szeibert, Erika Szabó, Géza Dallmann, Gábor Náray-Szabó, Bence Asbóth

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

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 Mg2+ as well as ternary complexes with bivalent metal ions and the open-chain inhibitor xylitol were crystallized for x-ray studies. We determined the crystal structures of the ternary complexes containing Mg2+, Mn2+, and Ca2+ at 2.2 to 2.5 Å resolutions, and refined them to R factors of 16.3, 16.6, and 19.1, respectively. We found that all metals are liganded by both engineered glutamates as well as by atoms O1 and O2 of the inhibitor. The similarity of the coordination of Ca2+ to that of the cofactors as well as results with Be2+ weaken the assumption that geometry differences should account for the catalytic noncompetence of this ion. Kinetic results of the D254E/D256E mutant enzyme showed that the significant decrease in Ca2+ inhibition was accompanied by a similar reduction in the enzymatic activity. Qualitative argumentation, based on the protein electrostatic potential, indicates that the proximity of the negative side chains to the substrate significantly reduces the electrostatic stabilization of the transition state. Furthermore, due to the smaller size of the catalytic metal site, no water molecule, coordinating the metal, could be observed in ternary complexes of the double mutant. Consequently, the proton shuttle step in the overall mechanism should differ from that in the wild type. These effects can account for the observed decrease in catalytic efficiency of the D254E/D256E mutant enzyme.

Original languageEnglish
Pages (from-to)183-193
Number of pages11
JournalProteins: Structure, Function and Genetics
Volume28
Issue number2
DOIs
Publication statusPublished - Jul 14 1997

Keywords

  • D-xylose isomerase
  • Enzyme kinetics
  • Protein crystallography

ASJC Scopus subject areas

  • Structural Biology
  • Biochemistry
  • Molecular Biology

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