Development and characterization of an immobilized enzyme reactor (IMER) based on human glyceraldehyde-3-phosphate dehydrogenase for on-line enzymatic studies.

Immobilized enzyme reactors (IMERs) for on-line enzymatic studies are useful tool to select specific inhibitors and may be used for direct determination of drug-receptor binding interactions and for the rapid on-line screening to identify specific inhibitors. This technique has been shown to increase the stability of enzymes. The enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays an important role in the life cycle of the Trypanosoma cruzi and it has become a key target in the drug discovery program for Chagas' disease. Crystallographic studies have indicated that there are significant inter-species differences in GAPDH activity and sensitivity. For example the active sites of GAPDH in T. cruzi and humans differ by a substitution of ASP(210) (T. cruzi) by Leu(194) in human. Based on this information we initiated the study to develop optimal conditions for the covalent immobilization of the human GAPDH enzyme on a modified capillary support (400 mm x 0.10 mm). The chromatographic separation of NAD from NADH was achieved using a RP-Spherex-diol-OH (10 cm x 0.46 cm, 10 microm, 100 A) column. By using multidimensional HPLC chromatography system it was possible to investigate the activity and kinetic parameters of the GAPDH-IMER. The values obtained for D-GA3P and NAD were K(m)=3.5+/-0.2 mM and 0.75+/-0.04 mM, respectively, and were compared with values obtained with the free enzyme. The activity of the immobilized GAPDH has been preserved for over 120 days.