Lee H J, Chang G G
Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan, Republic of China.
Biochem J. 1987 Jul 15;245(2):407-14. doi: 10.1042/bj2450407.
The structural requirements of the NADP+ molecule as a coenzyme in the oxidative decarboxylation reaction catalysed by pigeon liver malic enzyme were studied by kinetic and fluorimetric analyses with various NADP+ analogues and fragments. The substrate L-malate had little effect on the nucleotide binding. Etheno-NADP+, 3-acetylpyridine-adenine dinucleotide phosphate, and nicotinamide-hypoxanthine dinucleotide phosphate act as alternative coenzymes for the enzyme. Their kinetic parameters were similar to that of NADP+. Thionicotinamide-adenine dinucleotide phosphate, 3-aminopyridine-adenine dinucleotide phosphate, 5'-adenylyl imidodiphosphate, nicotinamide-adenine dinucleotide 3'-phosphate and NAD+ act as inhibitors for the enzyme. The first two were competitive with respect to NADP+ and non-competitive with respect to L-malate; the other inhibitors were non-competitive with NADP+. All NADP+ fragments were inhibitory to the enzyme, with a wide range of affinity, depending on the presence or absence of a 2'-phosphate group. Compounds with this group bind to the enzyme 2-3 orders of magnitude more tightly than those without this group. Only compounds with this group were competitive inhibitors with respect to NADP+. We conclude that the 2'-phosphate group is crucial for the nucleotide binding of this enzyme, whereas the carboxyamide carbonyl group of the nicotinamide moiety is important for the coenzyme activity. There is a strong synergistic effect between the binding of the nicotinamide and adenosine moieties of the nucleotide molecule.
通过使用各种NADP⁺类似物和片段进行动力学和荧光分析，研究了NADP⁺分子作为鸽肝苹果酸酶催化氧化脱羧反应中辅酶的结构要求。底物L-苹果酸对核苷酸结合影响很小。乙烯基-NADP⁺、3-乙酰吡啶-腺嘌呤二核苷酸磷酸和烟酰胺-次黄嘌呤二核苷酸磷酸可作为该酶的替代辅酶。它们的动力学参数与NADP⁺相似。硫代烟酰胺-腺嘌呤二核苷酸磷酸、3-氨基吡啶-腺嘌呤二核苷酸磷酸、5'-腺苷酰亚氨基二磷酸、烟酰胺-腺嘌呤二核苷酸3'-磷酸和NAD⁺可作为该酶的抑制剂。前两种抑制剂对NADP⁺具有竞争性，对L-苹果酸无竞争性；其他抑制剂对NADP⁺无竞争性。所有NADP⁺片段对该酶均有抑制作用，其亲和力范围广泛，这取决于2'-磷酸基团的存在与否。含有该基团的化合物与酶的结合比不含该基团的化合物紧密2-3个数量级。只有含有该基团的化合物对NADP⁺是竞争性抑制剂。我们得出结论，2'-磷酸基团对于该酶的核苷酸结合至关重要，而烟酰胺部分的羧酰胺羰基对于辅酶活性很重要。核苷酸分子的烟酰胺和腺苷部分的结合之间存在强烈的协同效应。
