Choi S Y, Bahn J H, Lee B R, Jeon S G, Jang J S, Kim C K, Jin L H, Kim K H, Park J S, Park J, Cho S W
Department of Genetic Engineering, Division of Life Sciences, Hallym University, Chunchon, Korea.
J Neurochem. 2001 Feb;76(3):919-25. doi: 10.1046/j.1471-4159.2001.00147.x.
An NAD+ dependent succinic semialdehyde dehydrogenase from bovine brain was inactivated by pyridoxal-5'- phosphate. Spectral evidence is presented to indicate that the inactivation proceeds through formation of a Schiff's base with amino groups of the enzyme. After NaBH(4) reduction of the pyridoxal-5'-phosphate inactivated enzyme, it was observed that 3.8 mol phosphopyridoxyl residues were incorporated/enzyme tetramer. The coenzyme, NAD+, protected the enzyme against inactivation by pyridoxal-5'-phosphate. The absorption spectrum of the reduced and dialyzed pyridoxal-5'-phosphate-inactivated enzyme showed a characteristic peak at 325 nm, which was absent in the spectrum of the native enzyme. The fluorescence spectrum of the pyridoxyl enzyme differs completely from that of the native enzyme. After tryptic digestion of the enzyme modified with pyridoxal-5'-phosphate followed by [3H]NaBH4 reduction, a radioactive peptide absorbing at 210 nm was isolated by reverse-phase HPLC. The sequences of the peptide containing the phosphopyridoxyllysine were clearly identical to sequences of other mammalian succinic semialdehyde dehydrogenase brain species including human. It is suggested that the catalytic function of succinic semialdehyde dehydrogenase is modulated by binding of pyridoxal-5'-phosphate to specific Lys(347) residue at or near the coenzyme-binding site of the protein.
来自牛脑的一种依赖NAD⁺的琥珀酸半醛脱氢酶被磷酸吡哆醛失活。提供的光谱证据表明，失活过程是通过与酶的氨基形成席夫碱进行的。在用NaBH₄还原磷酸吡哆醛失活的酶后，观察到每酶四聚体掺入3.8摩尔磷酸吡哆醛残基。辅酶NAD⁺可保护该酶免受磷酸吡哆醛的失活作用。还原并透析后的磷酸吡哆醛失活酶的吸收光谱在325nm处有一个特征峰，而天然酶的光谱中没有此峰。磷酸吡哆醛酶的荧光光谱与天然酶的荧光光谱完全不同。在用磷酸吡哆醛修饰该酶后进行胰蛋白酶消化，然后用[³H]NaBH₄还原，通过反相高效液相色谱法分离出一个在210nm处有吸收的放射性肽段。含有磷酸吡哆醛赖氨酸的肽段序列与包括人类在内的其他哺乳动物脑琥珀酸半醛脱氢酶的序列明显相同。有人提出，琥珀酸半醛脱氢酶的催化功能是通过磷酸吡哆醛与蛋白质辅酶结合位点处或附近的特定赖氨酸（347）残基结合来调节的。
