PMID: 9188736Jun 1, 1997Paper

Role of electrostatics at the catalytic metal binding site in xylose isomerase action: Ca(2+)-inhibition and metal competence in the double mutant D254E/D256E

Proteins
M FuxreiterB Asbóth

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 A 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 ...Continue Reading

References

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Citations

Jan 10, 2014·Protein Engineering, Design & Selection : PEDS·Mary Jo WaltmanAndrey Kovalevsky
Jun 14, 2014·The FEBS Journal·Ponnandy PrabhuJung-Kul Lee
Dec 6, 2011·Journal of Industrial Microbiology & Biotechnology·Hajer Ben HlimaSamir Bejar
Jan 11, 2001·Biochimica Et Biophysica Acta·B S HartleyM Rangarajan
Dec 26, 2001·Chemical Reviews·X HeH W Liu Hw

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