[Correlation of conformational change with enzyme activity of glucose isomerase in denaturants monitored with high performance liquid chromatography].
Glucose isomerase (GI) can catalyze in vitro the isomerization of D-glucose to D-fructose. So it is an extremely important industrial enzyme in the commercial conversion of starch to high fructose syrups. In the previous papers, we have purified and characterized the enzyme from streptomyces diastaticus M1033 of China and obtained the crystal structures by X-ray. In this paper, a method for measurement of the dynamic conformational change procedure of glucose isomerase in various concentrations of denaturants by HPLC has been established. At first the relative molecular mass of GI in solution is measured by HPLC on PROTEIN PAK 300SW (7.5 mm i.d. x 30 cm) column. The relative molecular mass of GI is about 150,000. So GI exists as tetramer in the solution without denaturants. In 0-5 mol/L guanidine hydrochloride, incubated at 30 degrees C for 30 min, GI is gradually dissociated into monomer, and at the same time its activity gradually disappears. In various concentrations of urea and incubation at 30 degrees C for 30 min (or 60 degrees C for 1 h), the results are different from that in guanidine, because the monomers peaks of GI is not found. Only in certain concentrations of urea, the small dimer peaks of GI is found, but the activity of GI significantly disappears. Moreover as the increase of the urea concentration, the retention time of tetramer peak is gradually decreases. From the fluorescence spectra, we found the conformation of GI changed in the solution of urea. So perhaps in urea, the conformation of GI become a little unfolded, and the active region is partly damaged, which makes GI partly inactive. Dissociation into inactive monomers and conformation partly unfolding are all the reason of GI inactivation in denaturants.
Wang
C
C
College of Life Sciences, University of Science and Technology of China, Hefei 230026, China.
Tao
L
L
Geng
L
L
Luo
D
D
Teng
M
M
Wang
Y
Y
Cui
T
T
D019747:Aldose-Ketose Isomerases / Q000737:chemistry / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013329:Structure-Activity Relationship
D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase