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Cha JH, Cho YJ, Whitaker RD, Carrell HL, Glusker JP, Karplus PA, Batt CA
Perturbing the Metal Site in D-Xylose Isomerase - Effect of Mutations of His-220 on Enzyme Stability
Journal of Biological Chemistry (1994) 269:2687-2694.
Abstract
The histidine residue at position 220 in the Streptomyces rubiginosus D-xylose isomerase is conserved in all D-xylose isomerases. The three-dimensional structure of D-xylose isomerase reveals that His-220 is part of the octahedral coordination sphere of M2, one of two metal ions (Mn2+) in the active site. This work describes the effects of replacing His- 220 with Ser, Glu, Asn, and Lys. The consequences of these amino acid substitutions on enzyme activity, thermostability, and structure were analyzed by kinetic, denaturation, and crystallographic methods. The k(cat) values for H220S, H220N, and H220E are only 0.3-0.5% of the wild-type values, and the K(m) for each of these mutant enzymes increased by 30-40-fold over the wild-type value. The mutant enzyme H220K did not exhibit any measurable activity. Thermal denaturation studies (T(m) values) indicate that the H220S and H220N mutant enzymes are approximately 58-degrees-C less stable than the wild-type enzyme, whereas H220E and H220K are 13-24-degrees-C less stable than the wild-type enzyme. To analyze the molecular basis for this decreased thermostability, the crystal structures of the H220S, H220N, and H220E mutant enzymes complexed with Mn2+ have been determined at 1.95, 1.90, and 1.75 angstrom, respectively. In the H220S structure, a water molecule effectively replaces the Nepsilon-2 atom of the imidazole ring of His-220 and mediates the interaction between Mn2+ at the M2 site and Ser- 220. A similar water-mediated interaction between the metal ion and Asn-220 is observed in H220N. No direct or water-mediated interactions between the carboxyl group of Glu-220 and the metal are observed in H220E. Whereas octahedral coordination is maintained for the metal at the M2 site in H220S and H220N, a pentahedral coordination with the metal at the M2 site is observed in H220E. Metal activation measurements support the observation that metal binding is perturbed and is responsible for thermal lability of His-220 mutants.
Note
Publication Date: 1994-01-28.
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