title: Role of the divalent metal ion in sugar binding, ring opening, and isomerization by D-xylose isomerase: replacement of a catalytic metal by an amino acid.
abstract: The distinct roles of the two magnesium ions essential to the activity of D-xylose isomerase from Streptomyces olivochromogenes were examined. The enzyme-magnesium complex was isolated, and the stoichiometry of cation binding determined by neutron activation analysis to be 2 mol of magnesium per mole of enzyme. A plot of Mg2+ added versus Mg2+ bound to enzyme is consistent with apparent KD values of < or = 0.5-1.0 mM for one Mg2+ and < or = 2-5 mM for the second. A site-directed mutant of D-xylose isomerase was designed to remove the tighter, tetracoordinated magnesium binding site (site 1, Mg-1); Glu180 was replaced with Lys180. The stoichiometry of metal binding to this mutant, E180K, is 1 mol of magnesium per mole of enzyme. Ring-opening assays with 1-thioglucose (H2S released upon ring opening) show E180K catalyzes the opening of the sugar ring at 20% the rate of the wild-type, but E180K does not catalyze isomerization of glucose to fructose. Thus, the magnesium bound to Glu180 is essential for isomerization but not essential for ring opening. The X-ray crystallographic structures of E180K in the absence of magnesium and in the presence and absence of 250 mM glucose were obtained to 1.8-A resolution and refined to R factors of 17.7% and 19.7%, respectively. The wild-type and both E180K structures show no significant structural differences, except the epsilon-amino group of Lys180, which occupies the position usually occupied by the Mg-1.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts 02254-9110.
authors.forename: K N
authors.initials: KN
authors.lastname: Allen
authors.forename: A
authors.initials: A
authors.lastname: Lavie
authors.forename: A
authors.initials: A
authors.lastname: Glasfeld
authors.forename: T N
authors.initials: TN
authors.lastname: Tanada
authors.forename: D P
authors.initials: DP
authors.lastname: Gerrity
authors.forename: S C
authors.initials: SC
authors.lastname: Carlson
authors.forename: G K
authors.initials: GK
authors.lastname: Farber
authors.forename: G A
authors.initials: GA
authors.lastname: Petsko
authors.forename: D
authors.initials: D
authors.lastname: Ringe
mesh_terms: D019747:Aldose-Ketose Isomerases; D000596:Amino Acids / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002238:Carbohydrate Epimerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D050260:Carbohydrate Metabolism; D002384:Catalysis; D002413:Cations, Divalent; D018360:Crystallography, X-Ray; D005971:Glutamates; D018698:Glutamic Acid; D008239:Lysine; D008274:Magnesium / Q000378:metabolism* / Q000494:pharmacology; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D013302:Streptomyces / Q000201:enzymology; D013329:Structure-Activity Relationship
chemical_list: D000596:Amino Acids; D002413:Cations, Divalent; D005971:Glutamates; D018698:Glutamic Acid; D002238:Carbohydrate Epimerases; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase; D008274:Magnesium; D008239:Lysine