Iminosugar glycosidase inhibitors: structural and thermodynamic dissection of the binding of isofagomine and 1-deoxynojirimycin to beta-glucosidases

The design and synthesis of transition-state mimics reflects the growing need both to understand enzymatic catalysis and to influence strategies for therapeutic intervention. Iminosugars are among the most potent inhibitors of glycosidases. Here, the binding of 1-deoxynojirimycin and (+)-isofagomine to the "family GH-1" beta-glucosidase of Thermotoga maritima is investigated by kinetic analysis, isothermal titration calorimetry, and X-ray crystallography. The binding of both of these iminosugar inhibitors is driven by a large and favorable enthalpy. The greater inhibitory power of isofagomine, relative to 1-deoxynojirimycin, however, resides in its significantly more favorable entropy; indeed the differing thermodynamic signatures of these inhibitors are further highlighted by the markedly different heat capacity values for binding. The pH dependence of catalysis and of inhibition suggests that the inhibitory species are protonated inhibitors bound to enzymes whose acid/base and nucleophile are ionized, while calorimetry indicates that one proton is released from the enzyme upon binding at the pH optimum of catalysis (pH 5.8). Given that these results contradict earlier proposals that the binding of racemic isofagomine to sweet almond beta-glucosidase was entropically driven (Bülow, A. et al. J. Am. Chem. Soc. 2000, 122, 8567-8568), we reinvestigated the binding of 1-deoxynojirimycin and isofagomine to the sweet almond enzyme. Calorimetry confirms that the binding of isofagomine to sweet almond beta-glucosidases is, as observed for the T. maritima enzyme, driven by a large favorable enthalpy. The crystallographic structures of the native T. maritima beta-glucosidase, and its complexes with isofagomine and 1-deoxynojirimycin, all at approximately 2.1 A resolution, reveal that additional ordering of bound solvent may present an entropic penalty to 1-deoxynojirimycin binding that does not penalize isofagomine.     

Underwater blast loading of sandwich beams: Regimes of behaviour

Finite element (FE) calculations are used to develop a comprehensive understanding of the dynamic response of sandwich beams subjected to underwater blast loading, including the effects of fluid–structure interaction. Design maps are constructed to show the regimes of behaviour over a broad range of loading intensity, sandwich panel geometry and material strength. Over the entire range of parameters investigated, the time-scale associated with the initial fluid–structure interaction phase up to the instant of first cavitation in the fluid is much smaller than the time-scales associated with the core compression and the bending/stretching responses of the sandwich beam. Consequently, this initial fluid–structure interaction phase decouples from the subsequent phases of response. Four regimes of behaviour exist: the period of sandwich core compression either couples or decouples with the period of the beam bending, and the core either densifies partially or fully. These regimes of behaviour are charted on maps using axes of blast impulse and core strength. The simulations indicate that continued loading by the fluid during the core compression phase and the beam bending/stretching phase cannot be neglected. Consequently, analyses that neglect full fluid–structure interaction during the structural responses provide only estimates of performance metrics such as back face deflection and reaction forces at the supports. The calculations here also indicate that appropriately designed sandwich beams undergo significantly smaller back face deflections and exert smaller support forces than monolithic beams of equal mass. The optimum transverse core strength is determined for minimizing the back face deflection or support reactions at a given blast impulse. Typically, the transverse core strength that minimizes back face deflection is 40% below the value that minimizes the support reaction. Moreover, the optimal core strength depends upon the level of blast impulse, with higher strength cores required for higher intensity blasts.