Inactivation of glyceraldehyde-3-phosphate dehydrogenase by ferrylmyoglobin.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was rapidly inactivated by ferrylmyoglobin (ferrylMb). FerrylMb rapidly reacts with the sulfhydryl group of protein. We therefore surmised that the cysteine residues of GAPDH react with ferrylMb. However, the amount of ferrylMb required 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 and tryptophane did not prevent the enzyme inactivation. However, adding cysteine, but not tryptophane and tyrosine, produced a maximum absorption at 580 nm, suggesting the formation of sulfmyoglobin through the reaction of ferrylMb with cysteine. Furthermore, three new bands of molecular weights 50, 55 and 100 kDa occurred on the sodium dodecyl sulfate (SDS)-polyacrylamide gel during the exposure of GAPDH to ferrylMb. Cysteine, but not tryptophane and tyrosine, inhibited the formation of the bands. Kinetic data indicated that the binding site of NAD, but not glyceraldehyde-3-phosphate (G3P), was damaged by ferrylMb. These results suggest that inactivation of GAPDH induced by ferrylMb is predominantly due to oxidation of the essential cysteine 149, and that NAD protects the active site from oxidative attack of ferrylMb.
Miura
T
T
Hokkaido College of Pharmacy, Otaru, Japan. miurotos@hit.ac.jp
Muraoka
S
S
Ogiso
T
T
D000596:Amino Acids / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000032:analysis / Q000037:antagonists & inhibitors*; D007700:Kinetics; D008786:Metmyoglobin / Q000378:metabolism / Q000494:pharmacology*; D009243:NAD / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar
D000596:Amino Acids; C028048:ferrylmyoglobin; D009243:NAD; D008786:Metmyoglobin; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases