Kempton J B, Withers S G
Department of Chemistry, University of British Columbia, Vancouver, Canada.
Biochemistry. 1992 Oct 20;31(41):9961-9. doi: 10.1021/bi00156a015.
The beta-glucosidase from Agrobacterium faecalis (previously Alcaligenes faecalis) has been subjected to a detailed kinetic investigation with a range of substrates to probe its specificity and mechanism. It has a relatively broad specificity for the substrate sugar moiety and exhibits a classical pH dependence for its kinetic parameters with three different substrates and an identical pH dependence for its inactivation by a mechanism-based inactivator, cyclophellitol. Measurement of kcat and Km values for a series of aryl glucoside substrates has allowed construction of a Bronsted plot, the concave-downward shape of which is consistent with the anticipated two-step mechanism involving a glucosyl-enzyme intermediate which is formed and hydrolyzed via oxocarbonium ion-like transition states. The slope of the leaving group-dependent portion of the Bronsted plot (beta 1g = -0.7) indicates a large degree of bond cleavage at the transition state. Secondary deuterium kinetic isotope effects measured for five different aryl glucosides are also consistent with this mechanism and further suggest that the transition state for formation of the glucosyl-enzyme intermediate, probed with the slower substrates for which kH/kD = 1.06, is more SN2-like than that for its hydrolysis (for which kH/kD = 1.11). Reasons for this difference are proposed, and values of Ki for several ground-state and transition-state analogue inhibitors are presented which support the concept of sp2-hybridized transition states.
来自粪产碱菌（以前称为粪产碱杆菌）的β-葡萄糖苷酶已针对一系列底物进行了详细的动力学研究，以探究其特异性和作用机制。它对底物糖部分具有相对较宽的特异性，并且对于三种不同底物的动力学参数表现出典型的pH依赖性，对于基于机制的失活剂环戊糖醇的失活也表现出相同pH依赖性。通过测量一系列芳基葡萄糖苷底物的kcat和Km值，构建了一个布朗斯特图，其向下凹的形状与预期的两步机制一致，该机制涉及通过氧鎓离子样过渡态形成和水解的葡萄糖基酶中间体。布朗斯特图中离去基团依赖性部分的斜率（β1g = -0.7）表明在过渡态存在大量的键断裂。对五种不同芳基葡萄糖苷测量的二级氘动力学同位素效应也与该机制一致，并进一步表明，对于kH/kD = 1.06的较慢底物所探测的葡萄糖基酶中间体形成的过渡态，比其水解的过渡态（kH/kD = 1.11）更类似于SN2。提出了这种差异的原因，并给出了几种基态和过渡态类似物抑制剂的Ki值，这些值支持sp2杂化过渡态的概念。
