Kosman D J, Ettinger M J, Bereman R D, Giordano R S
Biochemistry. 1977 Apr 19;16(8):1597-601. doi: 10.1021/bi00627a011.
Previous results indicate that a tryptophan residue(s) may interact with the sugar substrate and Cu(II) atom of galactose oxidase (Ettinger, M. J., and Kosman, D. J. (1974), Biochemistry 13, 1248). We now show that N-bromosuccinimide (NBS) reduces enzymatic activity to 2% as two tryptophans are oxidized; only four residues are easily oxidized in the holoenzyme. An enzymatic activity vs. number of residues oxidized profile suggests that this inactivation is probably associated with only one of the first 2 residues oxidized. There is no evidence for chain cleavage or modification of amino acids other than tryptophan. While substrate protection is not afforded by the sugar substrate, the activity-related tryptophan is placed within the active-site locus by spectral evidence. NBS oxidation of two tryptophans results in a marked diminution of the large copper optical-activity transition at 314 nm. Under some reaction conditions, a doubling of ellipticity in the 600-nm region of copper CD is also observed. The effects of the NBS oxidation on the CD spectra of galactose oxidase permit the assignment of the 314-nm CD band to a charge-transfer transition and the 229-nm extremum to a specific tryptophan contribution. The AZZ parameter from electron spin resonance spectra is also markedly reduced by the NBS oxidation. Moreover, while cyanide binds to the native enzyme without reducing the Cu(II) atom, cyanide rapidly reduces the Cu(II) atom to Cu(I) in the NBS-oxidized enzyme. These CD and ESR results are taken to suggest that one aspect of the inactivation by NBS oxidation may be a conversion of the pseudosquare planar copper complex in the native enzyme to a more distorted, towards tetrahedral, complex in the inactivated enzyme. Since the inactivation can be accomplished without affecting binding of the sugar substrate, tryptophan oxidation must affect catalysis per se.
先前的结果表明，一个或多个色氨酸残基可能与半乳糖氧化酶的糖底物和铜（II）原子相互作用（埃廷格，M. J.，和科斯曼，D. J.（1974年），《生物化学》13卷，第1248页）。我们现在表明，N-溴代琥珀酰亚胺（NBS）将酶活性降低至2%，此时两个色氨酸被氧化；在全酶中只有四个残基易于被氧化。酶活性与被氧化残基数量的关系曲线表明，这种失活可能仅与最初被氧化的两个残基中的一个有关。没有证据表明除色氨酸外的氨基酸发生了链断裂或修饰。虽然糖底物不能提供底物保护，但光谱证据表明与活性相关的色氨酸位于活性位点区域内。两个色氨酸的NBS氧化导致314nm处大的铜光学活性跃迁显著减弱。在某些反应条件下，还观察到铜圆二色光谱600nm区域的椭圆率加倍。NBS氧化对半乳糖氧化酶圆二色光谱的影响使得314nm的圆二色带可归属于电荷转移跃迁，229nm的极值可归属于特定色氨酸的贡献。电子自旋共振光谱的AZZ参数也因NBS氧化而显著降低。此外，虽然氰化物与天然酶结合而不还原铜（II）原子，但在NBS氧化的酶中，氰化物会迅速将铜（II）原子还原为铜（I）。这些圆二色光谱和电子自旋共振光谱结果表明，NBS氧化失活的一个方面可能是天然酶中的假方形平面铜配合物转化为失活酶中更扭曲的、接近四面体的配合物。由于失活可以在不影响糖底物结合的情况下完成，色氨酸氧化必定影响催化本身。
