Perturbing the metal site in D-xylose isomerase. Effect of mutations of His-220 on enzyme stability.

J Cha,Y Cho,R.D Whitaker,H.L Carrell,J.P Glusker,P.A Karplus,C.A Batt

doi:10.1016/s0021-9258(17)41998-3

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 kcat values H220S, H220N, and H220E are only 0.3-0.5% of the wild-type values, and the Km 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 (Tm values) indicate that the H220S and H220N mutant enzymes are approximately 5-8 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 A, respectively. In the H220S structure, a water molecule effectively replaces the N epsilon-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.

Highlights

The histidine residue a t position 220 in the Streptomy- D-Xylose isomerase catalyzes the isomerization of a-D-xylose ces rubiginosus D-xylose isomerase is conserved in all to a-D-xylulose as well as a-D-glucose to a-D-hCtOSe in the
H220N mutant enzymes are -5-8 "C less stable than the wild-type enzyme, whereas H220E and H220K are 1324 "C less stable than thewild-type enzyme.To analyze enzyme isolated fromStreptomyces and certain other bacteria is a tetramer composed of four identical subunits, each monomer witha molecular mass of 43 kDa
The crystal structureof D-xylose isomerase reveals that each monomer folds into a triose-phosphateisomerase-like barrel, with the active site located at the carboxyl-terminal end of the barrel (Carrelelt al., 1984, 1989; Henrick et al, 1987, 1989; Collyer and Blow, 1989; Farber et a l . , 1987, 1989; Jenkins et al, 1992; Whitlow et al, 1991)

Summary

Total observations

The x-ray diffraction data for H220N were measured on a Nicolet show weak D-xylose isomerase activity, the active enzyme fraction was XlOOA area detector mounted on a Rigaku RU-200 rotating anode genidentitied by SDS-polyacrylamide geel lectrophoresisanalysis from the erator using CUK, radiation and double focusing nickel mirrors. H220S or H220E and the wild-type enzyme.Other than changes at the Thermostability Measurements-Metal-free enzyme was prepared by NH, and COOH termini and changes within the active site, no signifiextensive dialysis at 4 "C against 10 IIIM TEA (pH 7.3) and 1m~ EDTA, cant structural changes (root mean square differences of

RESULTS

These water molecules serve to stabilize the local structure

Findings

DISCUSSION