Block C, Lohmann R D, Beyersmann D
Institut für Zellbiologie, Biochemie und Biotechnologie, Universität Bremen.
Biol Chem Hoppe Seyler. 1990 Dec;371(12):1145-52. doi: 10.1515/bchm3.1990.371.2.1145.
5-Aminolevulinate dehydratase from bovine liver requires Zn(II) for its activity and is inhibited by micromolecular concentrations of Pb(II). To elucidate the structure of the active site and its interactions between the active site and the metal binding site we labeled the active site for fluorescence studies and ESR spectroscopy. o-Phthalaldehyde reacted with active site lysyl and cysteinyl residues to form a fluorescent isoindole derivative. The fluorescence energy was independent of the deprivation of Zn(II) and of its substitution by the inhibitory Pb(II). For ESR-studies five iodoacetamide and four isothiocyanate pyrrolidine-N-oxyl derivatives with various spacer lengths were used to label the active site cysteinyl and lysyl residues, respectively. The ESR spectra of the modified enzyme preparations exhibited a significant immobilization of all labels, even with the longest spacers employed. Obviously the reactive cysteine is buried more than 12 A, and the active site lysine more than 11 A in a cleft of the enzyme structure. Zn(II) deprivation from the iodoacetamide spin-labeled enzyme caused a marked reversible increase in label mobility, whereas the Pb(II) substituted enzyme exhibited a smaller mobilization of the label. These results are interpreted by a model of the active site where the reactive cysteinyl and the lysyl side groups are close enough to be crosslinked by o-phthalaldehyde within a distance of 3 A. A structural role is assigned to Zn(II) in the enzyme, since Zn(II) deprivation does not alter the fluorescence of the isoindole derivative and increases the mobility of the cysteine-bound spin labels in the active site cleft.
来自牛肝的5-氨基乙酰丙酸脱水酶的活性需要Zn(II)，并且会受到微分子浓度的Pb(II)的抑制。为了阐明活性位点的结构及其与金属结合位点之间的相互作用，我们对活性位点进行标记以便进行荧光研究和电子自旋共振光谱分析。邻苯二甲醛与活性位点的赖氨酰和半胱氨酰残基反应，形成一种荧光异吲哚衍生物。荧光能量与Zn(II)的缺失及其被抑制性的Pb(II)取代无关。对于电子自旋共振研究，分别使用了五种具有不同间隔长度的碘乙酰胺和四种异硫氰酸酯吡咯烷-N-氧基衍生物来标记活性位点的半胱氨酰和赖氨酰残基。即使使用最长的间隔物，修饰酶制剂的电子自旋共振光谱也显示出所有标记物都有显著的固定化。显然，反应性半胱氨酸被深埋在酶结构的裂隙中超过12 Å，而活性位点赖氨酸则超过11 Å。从碘乙酰胺自旋标记的酶中去除Zn(II)会导致标记物流动性显著可逆增加，而被Pb(II)取代的酶则显示出较小的标记物流动性。这些结果通过活性位点模型来解释，其中反应性半胱氨酰和赖氨酰侧链足够接近，能够在3 Å的距离内被邻苯二甲醛交联。在该酶中，Zn(II)被赋予了一种结构作用，因为去除Zn(II)不会改变异吲哚衍生物的荧光，并且会增加活性位点裂隙中与半胱氨酸结合的自旋标记物的流动性。
