Enzyme-Substrate Structures and Binding Pathways

Project: Research project

Description

DESCRIPTION (provided by applicant): 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.
StatusFinished
Effective start/end date8/30/855/31/12

Funding

  • National Institutes of Health: $740,020.00
  • National Institutes of Health: $735,925.00
  • National Institutes of Health: $573,244.00
  • National Institutes of Health: $568,935.00
  • National Institutes of Health: $574,435.00
  • National Institutes of Health: $562,023.00
  • National Institutes of Health: $622,788.00

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Triose-Phosphate Isomerase
Catalysis
Enzymes
Ligands
Substrates
Proteins
Spectroscopy
Protein Tyrosine Phosphatases
Purine-Nucleoside Phosphorylase
Kinetics
Hypoxanthine Phosphoribosyltransferase
Vibrational spectroscopy
Lasers
Imaging techniques
Phosphates
Hydrolysis
Hydrogen bonds
Nuclear magnetic resonance
X ray diffraction
Alkaline Phosphatase

ASJC

  • Engineering(all)
  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)