Thomsen N K, Poulsen F M
Carlsberg Laboratorium, Kemisk Afdeling, Valby Copenhagen, Denmark.
J Mol Biol. 1993 Nov 5;234(1):234-41. doi: 10.1006/jmbi.1993.1577.
Hydrogen exchange reactions of amides in hen egg white lysozyme that are pH dependent and have a low energy of activation have been shown to be in accordance with a reaction mechanism in two steps, an equilibrium step and an exchange step. These results are not in agreement with the model, proposed by C.K. Woodward & B.D. Hilton, known as the penetration model. Therefore our results suggest that this model should be revised. The amide hydrogen/deuterium exchange rates in hen egg white lysozyme were measured at 4 degrees C, 10 degrees C, 15 degrees C and 25 degrees C at pH 7.0 by 1H nuclear magnetic resonance spectroscopy. Activation energies of the exchange reactions in the range from 20 kJ mol-1 to 333 kJ mol-1 were obtained for 32 of the 129 residues in the protein. The amides of lysozyme studied here could be divided into two groups, one group of amides are characterized by an observed amide exchange rate (ko) in the range 10(-4) to 10(-6) s-1, an equilibrium constant k1/k2 close to 10(-5), a low energy of activation (20 to 50 kJ mol-1) and a distance less than 6 A from solvent. The other group of amides are characterized by a ko less than 10(-6) s-1, a k1/k2 close to 10(-7), higher energies of activation (40 to 330 kJ mol-1) and a distance more than 4 A from solvent. In terms of structure the amides of the last group are from the core of the protein. They are typically involved in a hydrogen bond and form part of the secondary structure either as interior alpha-helices or central strands of beta-sheets. The first group consists of amides that are in the shell of the protein between the core and the surface. These amides are typically hydrogen bonded and involved in secondary structure such as external alpha-helices or outer strands of beta-sheets and turns.
已证明，鸡蛋清溶菌酶中酰胺的氢交换反应依赖于pH值且活化能较低，符合两步反应机制，即平衡步骤和交换步骤。这些结果与C.K.伍德沃德和B.D.希尔顿提出的称为渗透模型的模型不一致。因此，我们的结果表明该模型应予以修正。通过1H核磁共振光谱法，在pH 7.0、4℃、10℃、15℃和25℃下测量了鸡蛋清溶菌酶中酰胺氢/氘的交换速率。该蛋白质129个残基中的32个残基的交换反应活化能在20 kJ mol-1至333 kJ mol-1范围内。此处研究的溶菌酶酰胺可分为两组，一组酰胺的特征是观察到的酰胺交换速率（ko）在10(-4)至10(-6) s-1范围内，平衡常数k1/k2接近10(-5)，活化能较低（20至50 kJ mol-1），与溶剂的距离小于6 Å。另一组酰胺的特征是ko小于10(-6) s-1，k1/k2接近10(-7)，活化能较高（40至330 kJ mol-1），与溶剂的距离大于4 Å。就结构而言，最后一组的酰胺来自蛋白质的核心。它们通常参与氢键形成，并作为内部α螺旋或β折叠的中心链构成二级结构的一部分。第一组由位于蛋白质核心与表面之间外壳中的酰胺组成。这些酰胺通常形成氢键，并参与二级结构，如外部α螺旋或β折叠的外侧链和转角。
