Oxygen-18 leaving group kinetic isotope effects on the hydrolysis of nitrophenyl glycosides. 1. beta-galactosidease-catalyzed hydrolysis.
Oxygen-18 leaving group kinetic isotope effects on the hydrolysis of nitrophenyl glycosides. 1. beta-galactosidease-catalyzed hydrolysis.
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Oxygen-18 离去基团动力学同位素对硝基苯糖苷水解的影响。
DOI：
10.1021/bi00514a031
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发表时间：
1981
期刊：
影响因子：
2.9
通讯作者：
J. Kirsch
中科院分区：
文献类型：
--
作者：
S. Rosenberg;J. Kirsch
测定了β-半乳糖苷酶催化对-硝基苯基-β-D-半乳糖苷(I)和2，4-二硝基苯基-β-D-半乳糖苷(II)的Vmax(V)和Vmax/Km(V/K)的氧-18离基动力学同位素效应(Kies)。前者在V和V/K上的KI值分别为1.022+/-0.002和1.014+/-0.003，后者的KI值分别为1.002+/-0.0009和1.030+/-0.003。这些结果表明，在底物饱和时，键的断裂在很大程度上决定了I的速率，而不是II的速率。两种底物的第一个不可逆步骤必须包括断裂与硝基苯基离开基团的键。该反应的反应机理具有两条平行的底物水解途径。除了最具活性的底物外，所有底物的主要途径都包括酶对苷元的SN2亲核取代，以产生共价半乳糖基酶，该酶又通过水的亲核连接而被水解。最活跃的底物(例如，II)来自短暂的酶结合的半乳糖氧碳离子，该离子在酶之间分配以得到共价的半乳糖基酶和水以产生半乳糖。
Oxygen-18 leaving group kinetic isotope effects (KIEs) have been determined on both Vmax (V) and Vmax/Km (V/K) for the beta-galactosidase-catalyzed hydrolysis of p-nitrophenyl beta-D-galactoside (I) and 2,4-dinitrophenyl beta-D-galactoside (II). The former substrate exhibits KIEs of 1.022 +/- 0.002 and 1.014 +/- 0.003 on V and V/K, respectively, while corresponding KIEs for the latter are 1.002 +/- 0.0009 and 1.030 +/- 0.003. These results indicate that bond scission is largely rate determining for I but not for II at substrate saturation. The first irreversible step for both substrates must involve cleavage of the bond to the nitrophenyl leaving group. The mechanism proposed for this reaction is characterized by two parallel pathways for substrate hydrolysis. The predominant route for all but the most reactive substrates involves a SN2 nucleophilic displacement of aglycon by the enzyme to yield a covalent galactosyl-enzyme which in turn is hydrolyzed via a nucleophilic attach by water. The most reactive substrates (e.g., II) from transiently an enzyme-bound galactosyl oxo-carbonium ion which partitions between enzyme to give the covalent galactosyl-enzyme and H2O to yield galactose.