Abstract

To elucidate the structure-function relationships in glucose phosphate isomerase (GPI), we established an expression system for human GPI as a fusion protein with glutathione S-transferase (GST) in E. coli. The GST-GPI fusion protein showed affinities for the substrates glucose 6-phosphate (G6P) and fructose 6-phosphate (F6P) similar to those of the native enzyme purified from human red blood cells (RBC). We expressed GPI cDNAs with four distinct disease-causing mutations and examined their enzymatic characteristics. Although each mutation caused reduced thermal stability, an amino acid substitution Thr-5-->Ile (T5I) exhibited marked thermal instability, suggesting that the amino-terminal of GPI is important for enzymatic stability. Thr-224 seemed not to be an essential residue, since the amino acid substitution Thr-224-->Met (T224M) showed normal substrate affinity in spite of a slight decrease in both specific activity and thermostability. Gln-343 and Asp-539 have been shown to be in close proximity to the putative catalytic sites, and the present study showed that both Gln-343-->Arg (Q343R) and Asp-539-->Asn (D539N) caused impaired substrate affinity; Q343R showed high Km for both G6P and F6P, whereas D539N showed significantly decreased affinity only for F6P. These results suggest that not only reduced enzymatic stability but also impaired kinetics may disturb RBC metabolism of the GPI variants associated with hereditary hemolytic anemia.

Copyright 1998 The Blood Cells Foundation.