von Bahr-Lindström H, Jeck R, Woenckhaus C, Sohn S, Hempel J, Jörnvall H
Biochemistry. 1985 Oct 8;24(21):5847-51. doi: 10.1021/bi00342a023.
The mitochondrial isozyme of horse liver aldehyde dehydrogenase was labeled with brominated [5-(3-acetylpyridinio)pentyl]diphosphoadenosine. Specific labeling of a coenzyme binding region was proven by an enzymatic activity of the isozyme with the nonbrominated coenzyme derivative, optical properties of the complex, stoichiometry of incorporation, and protection against inactivation. A cysteine residue was selectively modified by the brominated coenzyme analogue and was identified in a 35-residue tryptic peptide. This cysteine residue corresponds to Cys-302 of the cytoplasmic isozyme and has earlier been implicated in disulfiram binding, confirming a position close to the active site. In contrast, the butyl homologue of the coenzyme analogue labels another residue of the mitochondrial isozyme. Thus, in the same isozyme, two residues are selectively reactive. They are concluded to be close together in the tertiary structure and to be close enough to the coenzyme binding site to be differentially labeled by coenzyme analogues differing only by a single methylene group.
马肝醛脱氢酶的线粒体同工酶用溴化的[5-(3-乙酰吡啶基)戊基]二磷酸腺苷进行标记。通过该同工酶与未溴化辅酶衍生物的酶活性、复合物的光学性质、掺入的化学计量以及防止失活等方面，证实了辅酶结合区域的特异性标记。一个半胱氨酸残基被溴化辅酶类似物选择性修饰，并在一个35个残基的胰蛋白酶肽段中被鉴定出来。这个半胱氨酸残基对应于细胞质同工酶的Cys-302，并且此前已被认为与双硫仑结合有关，这证实了其靠近活性位点的位置。相比之下，辅酶类似物的丁基同系物标记线粒体同工酶的另一个残基。因此，在同一同工酶中，两个残基具有选择性反应性。可以得出结论，它们在三级结构中靠得很近，并且距离辅酶结合位点足够近，以至于仅相差一个亚甲基的辅酶类似物能够对它们进行差异标记。
