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.
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)
Allen
K N
KN
Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts 02254-9110.
Lavie
A
A
Glasfeld
A
A
Tanada
T N
TN
Gerrity
D P
DP
Carlson
S C
SC
Farber
G K
GK
Petsko
G A
GA
Ringe
D
D
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
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