Characterization of the cupin-type phosphoglucose isomerase from the hyperthermophilic archaeon Thermococcus litoralis.
The gene encoding phosphoglucose isomerase was cloned from Thermococcus litoralis, and functionally expressed in Escherichia coli. The purified enzyme, a homodimer of 21.5 kDa subunits, was biochemically characterized. The inhibition constants for four competitive inhibitors were determined. The enzyme contained 1.25 mol Fe and 0.24 mol Zn per dimer. The activity was enhanced by the addition of Fe(2+), but inhibited by Zn(2+) and EDTA. Enzymes with mutations in conserved histidine and glutamate residues in their cupin motifs contained no metals, and showed large decreases in k(cat). The circular dichroism spectra of the mutant enzymes and the wild type enzyme were essentially the same but with slight differences.
Jeong
Jong-Jin
JJ
Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Fushinobu
Shinya
S
Ito
Sohei
S
Jeon
Beong-Sam
BS
Shoun
Hirofumi
H
Wakagi
Takayoshi
T
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D001665:Binding Sites / Q000235:genetics / Q000502:physiology; D001667:Binding, Competitive / Q000187:drug effects; D002384:Catalysis; D002614:Chelating Agents / Q000494:pharmacology; D002942:Circular Dichroism; D003001:Cloning, Molecular; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005956:Glucose-6-Phosphate Isomerase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics*; D007501:Iron / Q000737:chemistry / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D013329:Structure-Activity Relationship; D013696:Temperature; D019712:Thermococcus / Q000201:enzymology*; D015032:Zinc / Q000737:chemistry / Q000494:pharmacology
D019843:Archaeal Proteins; D002614:Chelating Agents; D004791:Enzyme Inhibitors; D007501:Iron; D005956:Glucose-6-Phosphate Isomerase; D015032:Zinc