There has been progress towards elucidating the mechanism of Escherichia coli glycerol kinase (EcGK) control by its allosteric effectors fructose-1,6-bisphosphate (FBP) and IIAglc (a member of the phosphoenolpyruvate:glycose phosphotransferase system). Determining the mechanism requires analysis of the interaction between these effectors and the substrates of EcGK. In this study, a structural and kinetic approach was used to determine inhibition by both the effectors. For this work, the use of fluorescence anisotropy to observe ligand binding was investigated. Also, a foundation was laid for future NMR experiments with EcGK. For fluorescence studies, E36C EcGK was labeled with fluorescein and tested for changes in anisotropy in the presence of different ligands. To ensure that E36C was an appropriate representative of wildtype protein, initial velocity, inhibition, and heterotropic coupling assays were performed. Groundwork for future NMR experiments required analyzing substitutions of the native EcGK cysteines by initial velocity and inhibition studies. By comparing wildtype enzyme and E36C (variant of wildtype with an engineered cysteine residue at position 36), it was found that E36C is a suitable substitute and was not drastically affected by labeling with fluorescein. Anisotropy values differed upon binding of different ligands and enabled titrations of the enzyme substrate complexes with both effectors to obtain dissociation constants. This supports using the stopped-flow method to assess the on- and off- rates of substrates and to obtain values for Q coupling. Furthermore, the results for FBP showed that inhibition by FBP is K-type (affects affinity) with respect to ATP and V-type (affects enzyme velocity) with respect to ADP. The findings presented also showed that native cysteine substitutions effect some of the catalytic and allosteric parameters of EcGK and would be powerful reporters for ligand binding in NMR. However, the enzymes are unstable and new protocols for protein isolation will need to be drafted.

Ce travail s’inscrit dans le domaine des cages moléculaires, édifices dont la géométrie définit unecavité tridimensionnelle capable de contenir d’autres entités.L’objectif de cette thèse est la synthèse de cages moléculaires covalentes possédant despropriétés d’encapsulation et une taille modulables. Les cages synthétisées avec succèsincorporent des porphyrines de zinc ou bases libres, ainsi que huit ligands triazoles périphériques.Des molécules ont pu être encapsulées par coordination aux porphyrines ou par interactions π, cequi démontre la capacité de la cage à s’adapter à l’invité grâce à sa flexibilité. La coordinationd’ions argent(I) permet de passer d’une conformation aplatie à une conformation ouverte. Lecaractère commutable de la cage a été démontré par décoordination de ces ions. Enfin, lacoordination d’ions argent(I) aux ligands de la cage augmente la stabilité de deux ligandsditopiques, le DABCO et la pipérazine, au sein de la cavité. Elle permet aussi l’accès de la cavité àdes molécules non encapsulées en l’absence d’argent(I). Ces résultats démontrent le contrôleallostérique de l’encapsulation de molécules par un stimulus chimique. / This work belongs to the field of molecular cages, hollow structures enclosing a three-dimensionalcavity able to encapsulate other molecules.The goal of this thesis include the synthesis of molecular covalent cages with a controllablecavity size able to perform switchable guest encapsulation. A DABCO-templated click reactionafforded three flexible covalent cages, endowed with two zinc(II) or free-base porphyrins and eightperipheral triazole ligands. Various guest molecules could be encapsulated by coordination toporphyrins or π-π interactions, in an induced-fit mechanism, probing the ability of the cage to adaptto the guest thanks to its flexibility. Coordination of silver(I) ions to the peripheral triazoles allow toswitch from a flattened to an open and locked conformation. Removal of the ions proved thecommutability of the cage. Finally, coordination of silver(I) ions to triazole ligands increase thestability of two ditopic ligands, DABCO and piperazine, inside the cavity. It also allowed the accessto the cavity of molecules that were not otherwise encapsulated. These results shows the allostericcontrol of guest encapsulation by a chemical stimulus.