Effects of inherited mutations on catalytic activity and structural stability of human glucose-6-phosphate isomerase expressed in Escherichia coli.
Glucose-6-phosphate isomerase (GPI), a homodimeric enzyme, catalyzes the interconversion between glucose-6-phosphate and fructose-6-phosphate. In mammals, it can also act as an autocrine motility factor, neuroleukin, and maturation factor. Deficiency of the enzymatic activity in red blood cells causes nonspherocytic hemolytic anemia in human. To gain a more complete understanding of the molecular basis for the hemolytic anemia due to the GPI-deficiency, the wild-type enzyme and sixteen genetic variants were expressed in Escherichia coli and functionally characterized. Conclusions are as follows: (1) mutations usually have negative influences on catalytic parameters, particularly k(cat), as well as structure stability; (2) mutations at or close to the active site, including R273H, H389R, and S278L, cause great damage to the catalytic function, yet those at distance can still reduce the magnitude of k(cat), despite lesser extents; (3) mutations decrease the enzyme tolerance to heat or SDS by mechanisms of decreasing packing efficiency (V101M, T195I, S278L, L487F, L339P, T375R, I525T), weakening network bonding (R75G, R347C, R347H, R472H, E495K), increasing water-accessible hydrophobic surface (R83W), and destabilizing the ternary structure (T195I, R347C, R347H, and I525T); (4) A300P, L339P, and E495K mutations may also negatively affect the protein folding efficiency.
Lin
Hua-Yang
HY
Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan 40227, Republic of China.
Kao
Ya-Heui
YH
Chen
Shu-Ting
ST
Meng
Menghsiao
M
D000595:Amino Acid Sequence; D000743:Anemia, Hemolytic / Q000201:enzymology / Q000235:genetics; D002384:Catalysis; D020134:Catalytic Domain; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D005956:Glucose-6-Phosphate Isomerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
D011994:Recombinant Proteins; D005956:Glucose-6-Phosphate Isomerase