Miura T, Muraoka S, Ogiso T
Hokkaido College of Pharmacy, Otaru, Japan.
Chem Biol Interact. 1997 Nov 28;107(3):173-83. doi: 10.1016/s0009-2797(97)00085-9.
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.
甘油醛-3-磷酸脱氢酶（GAPDH）可被高铁肌红蛋白（ferrylMb）迅速灭活。FerrylMb能快速与蛋白质的巯基反应。因此，我们推测GAPDH的半胱氨酸残基会与ferrylMb发生反应。然而，使该酶失活所需的ferrylMb量超过了酶中半胱氨酸的当量。FerrylMb不仅能被半胱氨酸还原，还能被酪氨酸和色氨酸还原。添加半胱氨酸可强烈阻止ferrylMb诱导的GAPDH失活，但添加酪氨酸和色氨酸并不能防止该酶失活。不过，添加半胱氨酸（而非色氨酸和酪氨酸）会在580nm处产生最大吸收峰，这表明ferrylMb与半胱氨酸反应形成了硫肌红蛋白。此外，在GAPDH与ferrylMb接触的过程中，十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶上出现了分子量分别为50、55和100kDa的三条新条带。半胱氨酸（而非色氨酸和酪氨酸）抑制了这些条带的形成。动力学数据表明，NAD的结合位点而非甘油醛-3-磷酸（G3P）的结合位点被ferrylMb破坏。这些结果表明，ferrylMb诱导的GAPDH失活主要是由于必需的半胱氨酸149被氧化，并且NAD可保护活性位点免受ferrylMb的氧化攻击。
