The long term objective of this application is to understand the molecular mechanisms by which allosteric ligands modify enzymatic behavior. Specifically, the allosteric properties of porcine heart fumarase, E. coli carbamoyl phosphate synthetase, bovine heart NAD-dependent isocitrate dehydrogenase, and rat liver phosphofructokinase will be investigated. Fumarase and carbamoyl phosphate synthetase are each regulated by allosteric modifiers that influence maximal velocity and/or Michaelis constants by altering the tertiary structure of the protein. The kinetic behavior of these enzymes will be studied using a thermodynamic linked-function approach. This approach provides for the quantitation of the dissociation constant of modifying ligand from free enzyme, the fractional influence that the modifying ligand has on maximal velocity, and the free energy of interaction between the substrate and allosteric ligand. These latter two parameters describe the efficacy of the allosteric ligand once bound. By observing the changes in these parameters produced by systematically altering the structure of the allosteric ligand or protein, we will be able to infer structural features of the ligand and protein important for these two separate facets of allosteric ligand action. This approach will be extended to investigate the mechanism of action of allosteric ligands that alter an enzyme's aggregation state as well and will be applied to a study of the regulation of isocitrate dehydrogenase by ADP, and phosphofructokinase by a wide variety of regulatory ligands. Isocitrate dehydrogenase should provide a straightforward example of the relationship between tertiary and quaternary levels of allosteric action, whereas phosphofructokinase represents a complex example of several different interdependent regulatory mechanisms. Despite this complexity, significant new insight should be obtained by quantitatively evaluating the pertinent free energy coupling parameters. This insight has been obscured in the past by the common assumption that the enzyme can exist in one of only two possible conformational states. This new insight is especially important in view of the vital role phosphofructokinase plays in carbohydrate homeostasis. The experimental methodologies required for these studies consist primarily of enzyme kinetics and fluorescence polarization measurements, the latter being performed on enzymes covalently labeled with a suitable extrinsic fluorescent probe.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM033216-03A1
Application #
3282618
Study Section
Biochemistry Study Section (BIO)
Project Start
1983-08-01
Project End
1989-06-30
Budget Start
1986-07-01
Budget End
1987-06-30
Support Year
3
Fiscal Year
1986
Total Cost
Indirect Cost
Name
University of Oklahoma Norman
Department
Type
Schools of Arts and Sciences
DUNS #
848348348
City
Norman
State
OK
Country
United States
Zip Code
73019
Whitaker, Amy M; Reinhart, Gregory D (2016) The effect of introducing small cavities on the allosteric inhibition of phosphofructokinase from Bacillus stearothermophilus. Arch Biochem Biophys 607:1-6
McGresham, Maria S; Reinhart, Gregory D (2015) Enhancing allosteric inhibition in Thermus thermophilus Phosphofructokinase. Biochemistry 54:952-8
McGresham, Maria S; Lovingshimer, Michelle; Reinhart, Gregory D (2014) Allosteric regulation in phosphofructokinase from the extreme thermophile Thermus thermophilus. Biochemistry 53:270-8
Ranjit, Suman; Dvornikov, Alexander; Holland, David A et al. (2014) Application of three-photon excitation FCS to the study of protein oligomerization. J Phys Chem B 118:14627-31
Mosser, Rockann; Reddy, Manchi C M; Bruning, John B et al. (2013) Redefining the role of the quaternary shift in Bacillus stearothermophilus phosphofructokinase. Biochemistry 52:5421-9
Mosser, Rockann; Reddy, Manchi C M; Bruning, John B et al. (2012) Structure of the apo form of Bacillus stearothermophilus phosphofructokinase. Biochemistry 51:769-75
Tie, Cuijuan; Reinhart, Gregory D (2012) An in vivo approach to isolating allosteric pathways using hybrid multimeric proteins. Methods Mol Biol 796:307-15
Wang, Shanzhi; Lasagna, Mauricio; Daubner, S Colette et al. (2011) Fluorescence spectroscopy as a probe of the effect of phosphorylation at serine 40 of tyrosine hydroxylase on the conformation of its regulatory domain. Biochemistry 50:2364-70
Bigley, Andrew N; Reinhart, Gregory D (2010) The N-terminus of glycogen phosphorylase b is not required for activation by adenosine 5'-monophosphate. Biochemistry 49:4760-5
Fenton, Aron W; Reinhart, Gregory D (2009) Disentangling the web of allosteric communication in a homotetramer: heterotropic inhibition in phosphofructokinase from Escherichia coli. Biochemistry 48:12323-8

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