The broad purpose of this work is to determine the structure and kinetics, and so 'image', protein and protein- ligand structural deformation and transformations that take place during enzyme catalyzed phosphoryl transfer reactions. Several spectroscopic methods, including difference FTIR and Raman spectroscopy, microfluidic mixing with IR and fluorescence detection, and IR and fluorescence temperature-jump relaxation spectroscopy will be used to detect the structures and structural evolution of the protein-ligand system at ultra high resolution and from ns to seconds time scales along the reaction pathway. This combination of advanced, novel techniques will allow us to visualize the sequence and timing of individual atomic-level events occurring in enzyme catalysis not possible through simple reaction monitoring or static structural pictures. Two enzyme systems, protein-tyrosine phosphatase (PTPase) and alkaline phosphatase (AP), will be studied. They catalyze the same phosphate monoesterase reaction but with quite different protein architectures and dynamics. Three broad issues are to be studied so as to understand how each enzyme brings together the necessary functional groups to achieve catalysis, to understand how their atomic level differences bring about similar functions, and to discern their dynamic differences. (i) We will determine how specific functional protein residues distort bound substrates along the reaction pathway. (ii) The dynamics of ligand binding will be determined. (iii) The timing of specific protein conformational changes related to critical proton transfer events and phosphate substrate distortions will be determined. In addition, we will probe the physical and dynamic properties that allow the same AP active site to catalyze secondary reactions (""""""""functional promiscuity""""""""). The value of these studies lies in several directions. In the most basic sense, this work will lead to a molecular understanding of mechanism in a crucial class of enzymes and will allow us to answer some of the compelling scientific questions such as: how do dynamic, conformationally fluctuating enzymes bring together the necessary functional groups to achieve catalysis? And what are the physical properties that allow the same AP active site to catalyze secondary reactions? In order to accomplish these goals, this work will continue to develop new methods aimed at determining protein structure and imaging the motion of atoms and molecular groups within folded proteins. Lastly, although not a direct objective, by understanding the dynamical nature of enzymes at an atomic level, our work can contribute to the rational design of pharmaceuticals.
This work develops and applies new advanced methods of imaging, or 'seeing', the structures of proteins and how they work, very much in the spirit of the development of, for example, magnetic resonance imaging methods developed in the last century. The fruits of this work can lead to more thoroughly understanding disease and then to the discovery of new drugs and methods as well as laboratory diagnostic methods.
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