Structure-reactivity relationships for beta-galactosidase (Escherichia coli, lac Z). 1. Brønsted parameters for cleavage of alkyl beta-D-galactopyranosides.
Structure-reactivity relationships for beta-galactosidase (Escherichia coli, lac Z). 1. Brønsted parameters for cleavage of alkyl beta-D-galactopyranosides.
复制标题
β-半乳糖苷酶（大肠杆菌，lac Z）的结构-反应性关系。
作者：
Richard,JP;Westerfeld,JG;Lin,S
1995年7月11日收到的修订版摘要：已经制备了七种取代的烷基pD-吡喃半乳糖苷1-OR，并显示其对γ 3-半乳糖苷酶（大肠杆菌，lac Z）催化水解的优异底物是公平的。在pH8.6条件下，酶与1-OR反应的一级速率常数k_3（s ~（-1））和二级速率常数k_3/Km（M_1s ~（-1））的Brpnsted参数分别为（fig）~*_3 =-0.49 ~0.13和（Agl-V ~（-1））~*_3 =-0.75 ~0.14。log Xm和烷基醇离去基团的pKd之间存在弱相关性，这归因于酶与烷氧基离去基团处吸电子卤素取代基之间的疏水相互作用使米氏络合物稳定。这些结合相互作用可能是生产性的，以kc/Km的值表示，也可能是非生产性的，以&3的值表示。负值的β 1%与糖苷键的内环裂解的酶催化不一致。的值/？烷基β-D-吡喃半乳糖苷的酶催化裂解的IG介于缩醛的自发（fig %-1.25）和特定酸催化（fig %-1.0）裂解所观察到的途径之间，因此这些途径被排除在酶催化反应之外。从酶中去除金属辅因子Mg 2+导致β-半乳糖苷酶催化的1-OR裂解的（fig）k3降低约0.2单位。对这一变化的解释尚不清楚。β-半乳糖苷酶催化反应的Brpnsted系数与必需催化残基参与1-OR糖苷键断裂的协调通用酸催化和/或通过与镁的相互作用稳定烷氧基氧上产生的负电荷是一致的。方案1示出了在保留构型的情况下催化糖基转移的第一步的工作模型，这是在检查溶菌酶-抑制剂复合物的X射线晶体结构之后首次提出的（Blake等人，1967年）。这一提议现在得到了缩醛裂解的非酶催化模型研究的广泛结果的支持（Fife，1975）。该反应机理是由在糖基上的贫醇盐离子离去基团的亲核取代催化的必要条件定义的。
Revised Manuscript Received July 11, 1995® abstract: Seven substituted alkyl pD-galactopyranosides 1-OR have been prepared and shown to be fair to excellent substrates for hydrolysis catalyzed by y3-galactosidase (Escherichia coli, lac Z). Brpnsted parameters of (fi\g)* 3=-0.49±0.13 and (Agl-V,,,=-0.75±0.14, respectively, were determined at pH8.6 for k3 (s-1), the first-order rate constant for cleavage of enzyme-bound 1-OR, and kcJKm (M_1 s-1), the second-order rate constant for reaction of the free enzyme and 1-OR. There is a weak correlation between log Xm andthe pKd of the alkyl alcohol leaving group, which is attributed to stabilization of the Michaelis complex by hydrophobic interactions between the enzyme and electron-withdrawing halogen substituents at the alkoxy leaving group. These binding interactions are probably both productive and expressed in the value of kcJKm and nonproductive and expressed in the value of &3. The negative values of ß\% are inconsistent with enzymatic catalysis of endocyclic cleavage of the glycosidic bond. The values of/? ig for enzyme-catalyzed cleavage of alkyl/3-D-galactopyranosides lie between those observed for the spontaneous (fi\g%—1.25) and specific-acid-catalyzed (fi\g% 0) cleavage of acetals, and these pathways are therefore excluded for the enzyme-catalyzed reaction. Removal of the metal cofactor Mg2+ from the enzyme causes a~ 0.2 unit decrease in (fi\g) k3 for/3-galactosidase-catalyzed cleavage of 1-OR. The interpretation of this change in ß\$ is unclear. The Brpnsted coefficients for the/3-galactosidase-catalyzed reaction are consistent with participation by an essential catalytic residue in concerted general-acid catalysis of cleavage of the glycosidic bond of 1-OR and/or stabilization of developing negativecharge at the alkoxy oxygen by interaction with the magnesiumMany of the details of the mechanism for enzymatic catalysis of glycosyl transfer reactions are not well under-stood. Scheme 1 shows a working model for the first step for catalysis of glycosyl transfer with retention of configuration, which was first proposed after examination of an X-ray crystal structure of a lysozyme—inhibitor complex (Blake et al., 1967). This proposal is now supported by extensive results of model studies of nonenzymatic catalysis of acetal cleavage (Fife, 1975). This reaction mechanism is defined by the imperatives for catalysis of nucleophilic substitution of a poor alkoxide ion leaving group at glyco-