Phosphofructokinase controls the rate-limiting step of glycolysis which matches cellular demand for nutrients and energy under normal and stressful conditions. This project focuses on two aspects on PFK enzymology: the allosteric control of rabbit muscle PFK (RMPFK), and the allosteric differences between two bacterial PFKs. (1) expression of RMPFK is DF1020 E. coli hosts and purification of the cloned RMPFK: In order to avoid EcPFK-1 activity, RMPFK cDNA will be subcloned in a pPL2 (lambda pL) vector and transformed in E.coli cells deficient in EcPFK-1 activity. The cloned RMPFK will be expressed and purified using a newly-developed protocol. As a long term goal, purification of cloned RMPFK could lead to crystallographic studies on this enzyme. (2) Site-directed mutagenesis of RMPFK and properties of mutated proteins: Kunkel's method will be applied to create point and deletion mutations at predetermined sites. Sites for mutagenesis will be selected on the basis of the sequence homologies between RMPFK and bacterial PFKs and the crystal structures of the latter. Mutated RMPFK will be analyzed for kinetics, binding of substrates and effectors as well as submit interaction. A truncated PFK subunit lacking 31 amino acids (280-311) was found to exist naturally in human muscle and other tissue. The role of this polypeptide is entirely unknown. A mutant lacking these 31 amino acids will be constructed, expressed and analyzed for enzymatic activity, allosteric properties, its association with wild-type muscle PFK subunits and its effects on general muscle PFK enzymology. (3) Structural basis for the allosteric differences of EcPFK-1 and BsPFK: EcPFK-1 binds F6P cooperatively but BsPFK gives a hyperbolic profile vs {F6p}. Details about the T and R states of the PFKs are known from their crystal structures. Site-directed mutagenesis will be used to analyzed the residues involved in the allosteric transition and to identify those responsible for the allosteric differences of the two PFKs. (4) Stereospecificity at F6P and F2,6BP sites: The stereospecificity of F6p site of cloned BsPFK and certain mutants thereof will be studies to ascertain the exact residues involve din binding and to gain insight into the evolutionary changes that this site underwent. Moreover, the residues involve din the F2,6BP allosteric site will be investigated using certain mutant of RMPFK. In all cases, a series of structurally locked analogues of F6P and F2,6BP will be used and the kinetic data will be correlated with biophysical studies, i.e., direct binding, light scattering, UV and CD spectrometry.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK031676-08
Application #
3230262
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1983-07-01
Project End
1994-06-30
Budget Start
1992-07-01
Budget End
1993-06-30
Support Year
8
Fiscal Year
1992
Total Cost
Indirect Cost
Name
Louisiana State University A&M Col Baton Rouge
Department
Type
Schools of Arts and Sciences
DUNS #
075050765
City
Baton Rouge
State
LA
Country
United States
Zip Code
70803
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Zhu, X; Byrnes, M; Nelson, J W et al. (1995) Role of glycine 212 in the allosteric behavior of phosphofructokinase from Bacillus stearothermophilus. Biochemistry 34:2560-5
Auzat, I; Byrnes, W M; Garel, J R et al. (1995) Role of residue 161 in the allosteric transitions of two bacterial phosphofructokinases. Biochemistry 34:7062-8
Byrnes, M; Zhu, X; Younathan, E S et al. (1994) Kinetic characteristics of phosphofructokinase from Bacillus stearothermophilus: MgATP nonallosterically inhibits the enzyme. Biochemistry 33:3424-31
Li, J; Zhu, X; Byrnes, M et al. (1993) Site-directed mutagenesis of rabbit muscle phosphofructokinase cDNA. Mutations at glutamine 200 affect the allosteric properties of the enzyme. J Biol Chem 268:24599-606
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Li, J; Chen, Z; Lu, L et al. (1990) Sequence diversity in the 5' untranslated region of rabbit muscle phosphofructokinase mRNA. Biochem Biophys Res Commun 170:1056-60
Watkins, S F; Kim, S J; Fronczek, F R et al. (1990) The X-ray crystal structure of 2,3:4,5-di-O-isopropylidene-1-O-methyl-beta-D-fructopyranose. Carbohydr Res 197:33-40
Valdez, B C; French, B A; Younathan, E S et al. (1989) Site-directed mutagenesis in Bacillus stearothermophilus fructose-6-phosphate 1-kinase. Mutation at the substrate-binding site affects allosteric behavior. J Biol Chem 264:131-5

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