Enzyme kinetics and molecular modeling studies of G6PD_Mahidol associated with acute hemolytic anemia

Abstract

G6PD_Mahidol enzyme is the most common variant in the Achang Chinese ethnic group and clinically manifests as class II. In this study, G6PD_Mahidol enzyme was characterized by molecular modeling to understand its kinetics. G6PD_Mahidol, G6PD^G487A and G6PD^WT proteins were heterologously expressed in the G6PD-deficient DF213<i style=""> E. coli</i> strain, purified and their steady-state kinetic parameters were determined. Compared with G6PD^WT, the <i style="">K_m</i> and <i style="">V_max</i> of NADP⁺with G6PD^G487Awere about 28-fold and 12-fold lower, respectively. The <i style="">K_i</i>values of dehydroepiandrosterone (DHEA), NADPH and ATP with G6PD^G487A showed 29.5-fold, 2.36-fold reduction and 1.83-fold increase, respectively. A molecular modeling of G6PD^G487Awas performed based on the X-ray structure of human G6PD (PDB: 2BH9). It is suggested that Ser-163 might affect the stability of G6PD^G487A <img src='/image/spc_char/alpha.gif' border=0>-helix d and <img src='/image/spc_char/beta.gif' border=0>-strand E, besides the conformation of <img src='/image/spc_char/beta.gif' border=0>-strand D. In conclusion, the biochemical and structural properties of G6PD^G487Aand G6PD^WT enzymes are significantly different, which may be responsible for clinical diversity of G6PD deficiencies.