A regulatory motive of fundamental importance to metabolic control is the allosteric modification of enzymatic activity by metabolites. The long-term goal of this research program is to understand the molecular basis for allosteric regulation of enzyme activity. In particular we are interested in systems in which the allosteric ligands achieve their effects by altering the affinity of the enzyme for its substrate. Phosphofructokinase(PFK) from E. coli and B. stearothermophilus will be investigated as model systems. These enzymes are homotetramers containing a single active site and a single allosteric site per subunit. Despite this relatively simple composition, 10 unique pair-wise allosteric interactions can be identified. Hybrid forms of these enzymes have been produced that isolate individual allosteric interactions that collectively act to produce the regulatory properties of the tetramic enzyme. We propose to investigate the molecular basis for the allosteric properties displayed by each of these hybrids because of the defined nature of the functional interaction in each case. Improvements in the procedures used to produce these hybrids will be developed by creating an expression strain of E. coil that does not have either gene for PFK and by refining a procedure for producing hybrids in vivo by co-expressing both parent proteins in the same cell. A strategy is proposed for isolating each 2:2 hybrid that will allow for the isolation of each unique homotropic interaction and for the demonstration of how multiple interactions combine within a dimeric theoretical framework. The putative conduit by which allosteric influence is transmitted in each hybrid displaying a single interaction will be located through the systematic use of site-directed mutagenesis. Concurrently the structural basis for the allosteric communication will be studied with X-ray crystallography and time-resolved fluorescence spectroscopy. The latter technique should produce information regarding the dynamic properties of the protein that will complement the structural information obtained from crystallography. Finally PFK from both E. coil and B. stearothermophilus will be carefully compared with respect to how substrate and allosteric ligand analogs trigger their allosteric behavior. These enzymes differ in the apparent thermodynamic basis for their allosteric properties, a difference that might be related to differences in binding specificity.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM033216-22
Application #
7086437
Study Section
Biochemistry Study Section (BIO)
Program Officer
Ikeda, Richard A
Project Start
1983-08-01
Project End
2008-11-30
Budget Start
2006-07-01
Budget End
2008-11-30
Support Year
22
Fiscal Year
2006
Total Cost
$373,318
Indirect Cost
Name
Texas A&M University
Department
Biochemistry
Type
Schools of Earth Sciences/Natur
DUNS #
078592789
City
College Station
State
TX
Country
United States
Zip Code
77845
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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