INACTIVATION OF GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE BY FERRYLMYOGLOBIN

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was rapidly inactivat ed by ferrylmyoglobin (ferrylMb). FerrylMb rapidly reacts with the sul fhydryl group of protein. We therefore surmised that the cysteine resi dues of GAPDH react with ferrylMb. However, the amount of ferrylMb req uired to inactivate the enzyme was in excess of the equivalent amount of cysteine in the enzyme. FerrylMb was reduced not only by cysteine, but also by tyrosine and tryptophane. Adding cysteine strongly blocked the inactivation of GAPDH induced by ferrylMb, but adding tyrosine an d tryptophane did not prevent the enzyme inactivation. However, adding cysteine, but not tryptophane and tyrosine, produced a maximum absorp tion at 580 nm, suggesting the formation of sulfmyoglobin through the reaction of ferrylMb with cysteine. Furthermore, three new bands of mo lecular weights 50, 55 and 100 kDa occurred on the sodium dodecyl sulf ate (SDS)-polyacrylamide gel during the exposure of GAPDH to ferrylMb. Cysteine, but not tryptophane and tyrosine, inhibited the formation o f the bands. Kinetic data indicated that the binding site of NAD, but not glyceraldehyde-3-phosphate (G3P), was damaged by ferrylMb. These r esults suggest that inactivation of GAPDH induced by ferrylMb is predo minantly due to oxidation of the essential cysteine 149, and that NAD protects the active site from oxidative attack of ferrylMb. (C) 1997 E lsevier Science Ireland Ltd.