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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Biomedical Imaging and Bioengineering (NIBIB)
Type
Research Project (R01)
Project #
5R01EB001958-26
Application #
7847530
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Conroy, Richard
Project Start
1985-08-30
Project End
2012-05-31
Budget Start
2010-06-01
Budget End
2012-05-31
Support Year
26
Fiscal Year
2010
Total Cost
$740,020
Indirect Cost
Name
Albert Einstein College of Medicine
Department
Biochemistry
Type
Schools of Medicine
DUNS #
110521739
City
Bronx
State
NY
Country
United States
Zip Code
10461
Nie, Beining; Lodewyks, Kara; Deng, Hua et al. (2016) Active-Loop Dynamics within the Michaelis Complex of Lactate Dehydrogenase from Bacillus stearothermophilus. Biochemistry 55:3803-14
Deng, Hua (2013) Enzyme active site interactions by Raman/FTIR, NMR, and ab initio calculations. Adv Protein Chem Struct Biol 93:153-82
Ke, Shan; Ho, Meng-Chiao; Zhadin, Nickolay et al. (2012) Investigation of catalytic loop structure, dynamics, and function relationship of Yersinia protein tyrosine phosphatase by temperature-jump relaxation spectroscopy and X-ray structural determination. J Phys Chem B 116:6166-76
Andrews, Logan D; Deng, Hua; Herschlag, Daniel (2011) Isotope-edited FTIR of alkaline phosphatase resolves paradoxical ligand binding properties and suggests a role for ground-state destabilization. J Am Chem Soc 133:11621-31
Deng, Hua; Vu, Dung V; Clinch, Keith et al. (2011) Conformational heterogeneity within the Michaelis complex of lactate dehydrogenase. J Phys Chem B 115:7670-8
Zhadin, Nickolay; Callender, Robert (2011) Effect of osmolytes on protein dynamics in the lactate dehydrogenase-catalyzed reaction. Biochemistry 50:1582-9
Deng, Hua; Callender, Robert; Schramm, Vern L et al. (2010) Pyrophosphate activation in hypoxanthine--guanine phosphoribosyltransferase with transition state analogue. Biochemistry 49:2705-14
Zhang, Yong; Deng, Hua; Schramm, Vern L (2010) Leaving group activation and pyrophosphate ionic state at the catalytic site of Plasmodium falciparum orotate phosphoribosyltransferase. J Am Chem Soc 132:17023-31
Juszczak, Laura J; Desamero, Ruel Z B (2009) Extension of the tryptophan chi2,1 dihedral angle-W3 band frequency relationship to a full rotation: correlations and caveats. Biochemistry 48:2777-87
Deng, Hua; Brewer, Scott; Vu, Dung M et al. (2008) On the pathway of forming enzymatically productive ligand-protein complexes in lactate dehydrogenase. Biophys J 95:804-13

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