el Kebbaj M S, Latruffe N, Gaudemer Y
Biochim Biophys Acta. 1984 Sep 25;789(3):278-84. doi: 10.1016/0167-4838(84)90183-3.
D-beta-Hydroxybutyrate dehydrogenase D-3-hydroxybutyrate: NAD+ oxidoreductase, EC 1.1.1.30), a phosphatidylcholine-requiring enzyme, was irreversibly inactivated by a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) or a hydrophobic carbodiimide, N,N'-dicyclohexylcarbodiimide (DCCD). The inactivation is pseudo-first-order with a kinetic stoichiometry of about 1. Phospholipid-free apoenzyme was more sensitive towards these reagents than reconstituted phospholipid-enzyme or membrane-bound enzyme forms. Reduced coenzyme (NADH) protected the enzyme against the inactivation, while oxidized coenzyme (NAD+) in presence of 2-methylmalonate (a pseudo-substrate) gave a better protection. It was found that the phospholipid-free apoenzyme bound about 1 mol [14C]DCCD. This incorporation was prevented by EDAC, indicating that both reagents react at the same site. [14C]Glycine ethyl ester, a nucleophilic compound which reacts specifically with the carboxylcarbodiimide derivative was incorporated to the enzyme (1 mol [14C]glycine ethyl ester per polypeptide chain), whatever its form, in the presence of DCCD or EDAC. These results indicate the presence of one carboxyl group probably located at or near the coenzyme-binding site and near the interacting domain of the enzyme with phospholipid.
D-β-羟基丁酸脱氢酶（D-3-羟基丁酸：NAD⁺氧化还原酶，EC 1.1.1.30）是一种需要磷脂酰胆碱的酶，可被水溶性碳二亚胺1-乙基-3-(3-二甲基氨基丙基)碳二亚胺（EDAC）或疏水性碳二亚胺N,N'-二环己基碳二亚胺（DCCD）不可逆地失活。失活为假一级反应，动力学化学计量比约为1。无磷脂的脱辅基酶对这些试剂比重构的磷脂-酶或膜结合酶形式更敏感。还原型辅酶（NADH）可保护该酶免于失活，而在2-甲基丙二酸（一种假底物）存在下的氧化型辅酶（NAD⁺）提供了更好的保护。发现无磷脂的脱辅基酶结合了约1摩尔[¹⁴C]DCCD。EDAC可阻止这种结合，表明两种试剂在同一部位反应。无论酶为何种形式，在DCCD或EDAC存在下，[¹⁴C]甘氨酸乙酯（一种与羧基碳二亚胺衍生物特异性反应的亲核化合物）都可掺入该酶（每条多肽链1摩尔[¹⁴C]甘氨酸乙酯）。这些结果表明存在一个羧基，其可能位于辅酶结合位点处或附近以及酶与磷脂相互作用的结构域附近。
