Biophysical Investigations of Allostery in E.coli Adenylate Kinase Using Entropy Enhancing Mutations

Allostery;E.coli Adenylate Kinase;Microcalorimetry;NMR;Enzyme kinetics;Biophysics

Saavedra Espinoza, Harry G.Garcia-Moreno, Bertrand E. ;

Johns Hopkins University

关键词: Allostery;    E.coli Adenylate Kinase;    Microcalorimetry;    NMR;    Enzyme kinetics;    Biophysics;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/39520/SAAVEDRAESPINOZA-DISSERTATION-2015.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

Allostery is the regulatory effect that a perturbation in one region has on anotherdistal site of the same macromolecule. Although allostery has been studiedextensively, understanding of how a perturbation in one region can affect otherregion(s) is still incomplete. For a long time, allostery has been regarded as astructural-based phenomenon. However, it is currently known that allostery canalso be manifested without perturbation in the crystal structure. Also, it hasbeen demonstrated from studies of thermal adapted enzymes, that enzyme functionregulation can be promoted by increasing the conformational flexibility inregions other than the active site. To understand these different facets of allostery,we used Escherichia coli adenylate kinase as a model system. This enzyme iscomposed of three distinct domains: The LID, CORE and AMPbd domains. Toanswer the question of how conformational perturbations regulate enzyme function,entropy-enhancing Gly substitutions were used on distal uncharged solventexposed residues located far from the active site. Mutations on the three domainswere chosen in order to avoid or minimize structural distortions. Subsequently,the impact of increased conformational flexibility on the ensemble, bindingaffinity and catalytic activity were investigated with DSC, ITC and catalyticactivity assays. In addition, ensemble modulation was characterized by combiningHX-NMR and ITC techniques. Together, the results show that enzymes canuse local unfolding in regions distal to the active site to regulate macromolecularfunction. This regulation arises predominantly from the redistribution ofthe probabilities of states promoted by local destabilization of the enzyme. Also,this work indicates that allostery can also be understood from a thermodynamicpoint of view. Finally, it is shown that the LID interacts with the other domains.