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.
Showing the most recent 10 out of 12 publications
