Inactivation and conformation changes of the glycated and non-glycated D-glyceraldehyde-3-phosphate dehydrogenase during guanidine-HCl denaturation.

The glycated D-glyceraldehyde-3-phosphate dehydrogenases have been isolated from rabbit muscle and erythrocytes (He et al. (1995) Biochem. J. 309, 133-139). The circular dichroism spectrum in the near-ultraviolet of gGAPDH was different from that of GAPDH. Changes in intrinsic protein fluorescence and in the 410 nm fluorescence of the NAD derivatives introduced at the active sites of both the glycated and non-glycated GAPDH from rabbit were compared on inactivation during denaturation in GuHCl. Complete inactivation for the non-glycated enzyme occurred in 0.5 M GuHCl solution, however, that for the glycated enzyme occurred in the 0.35 M solution. The kinetic inactivation of gGAPDH was a biphasic process (the fast and slow phases). The fast phase for gGAPDH was faster than that of GAPDH. The kinetic exposure of the fluorescent NAD derivatives at the active sites of both enzymes was also biphasic with fast phase rates which approach those of the inactivation. It appears that glycation of the enzyme may disturb the spatial geometry of the functional groups responsible for the catalytic mechanism and affect the activity.