Giovannini P P, Rippa M, Dallocchio F, Tetaud M, Barrett M P, Hanau S
Department of Biochemistry and Molecular Biology, University, Ferrara, Italy.
Biochem Mol Biol Int. 1997 Sep;43(1):153-60. doi: 10.1080/15216549700203921.
o-phthalaldehyde inactivates homodimeric, NADP+ dependent, 6-phosphogluconate dehydrogenase from sheep liver, upon formation of a single isoindole derivative per enzyme subunit. This indicates that the thiol group of a cysteine residue or the epsilon-amino group of a lysine residue located within 3 A and crosslinked by the reagent is essential for catalysis. Fluorescence analyses of the modified enzyme suggest that the isoindole derivative forms at the binding site of the nicotinamide moiety of NADP+. The enzymes from Trypanosoma brucei and Lactococcus lactis are also inactivated suggesting a similar three-dimensional structure in this domain. The isoindole derivative does not form with two mutants of the T. brucei enzyme (Lys185His and Lys185Leu), this allowing to identify not only the lysine but also the cysteine involved in the cross-linking. The formation of the isoindole derivative inactivates not only the oxidative decarboxylation, but also two partial reactions catalysed by the enzyme.
邻苯二甲醛使来自绵羊肝脏的同二聚体、依赖NADP⁺的6-磷酸葡萄糖酸脱氢酶失活，每个酶亚基形成一个异吲哚衍生物。这表明位于3埃范围内并通过该试剂交联的半胱氨酸残基的巯基或赖氨酸残基的ε-氨基对于催化至关重要。对修饰酶的荧光分析表明，异吲哚衍生物在NADP⁺烟酰胺部分的结合位点形成。来自布氏锥虫和乳酸乳球菌的酶也被失活，表明该结构域具有相似的三维结构。异吲哚衍生物不会与布氏锥虫酶的两个突变体（Lys185His和Lys185Leu）形成，这不仅可以鉴定参与交联的赖氨酸，还可以鉴定半胱氨酸。异吲哚衍生物的形成不仅使氧化脱羧失活，还使该酶催化的两个部分反应失活。
