The purpose of this work is to understand the dynamics of enzymatic catalysis at a molecular level. Structure is probed with vibrational spectroscopic tools that are capable of determining the Raman and IR spectra of bound substrates and specific protein molecular moieties embedded in large proteins. Vibrational spectroscopy yields a very high resolution of structure (better than 0.01 A), and such resolution is key to understanding enzymatic catalysis. Also, we have recently developed kinetic approaches, principally laser induced temperature relaxation spectroscopy, that can measure molecular motions in proteins over a broad time range (10 ps to minutes, some 13 orders of magnitude). These mechanistically important motions have been largely unstudied because of here-to-fore technical limitations. The kinetics of ligand binding/release will be probed here. Specific enzyme systems are studied for their scientific importance and biomedical relevance. Extensive studies are to be performed on triosephosphate isomerase (TIM). Our studies will characterize the strengths of putative compressed hydrogen bonds at the active site and characterize the enediol(ate) intermediate(s). Structure/function relationships will be formulated through measurements of a series of kinetically characterized active site mutants. The motion(s) of TIM's catalytically important mobile loop in releasing bound ligands and the time course of TIM-substrate to TIM-product inner complex conversion will be investigated by laser induced T-jump relaxation spectroscopy over our time range. This work is in a close collaboration with the laboratory of Prof. Ann McDermott, which will be performing high resolution (better than 1.2/k) X-ray diffraction work and NMR structural and dynamics studies of TIM in parallel to our studies. We shall continue our studies of the molecular mechanism(s) of phosphate hydrolysis and phosphoryl transfer in several systems: the protein-tyrosine phosphatases (PTPases), purine nucleoside phosphorylase (PNP), and hypoxanthine-guanine (xanthine) phosphoribosyltransferases (HG(X)PRT). High resolution vibrational structural studies will be performed to determine the electronic nature of the reaction pathway and to dissect the role of specific active site molecular factors bringing about catalysis. The dynamics of loop motion and its relationship to ligand release and catalysis will be studied in PTP and HGPRT. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of Biomedical Imaging and Bioengineering (NIBIB)
Type
Research Project (R01)
Project #
5R01EB001958-24
Application #
7264562
Study Section
Special Emphasis Panel (ZRG1-SSS-B (01))
Program Officer
Zhang, Yantian
Project Start
1985-08-30
Project End
2009-05-31
Budget Start
2007-08-01
Budget End
2009-05-31
Support Year
24
Fiscal Year
2007
Total Cost
$568,935
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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