Site-Directed Mutagenesis in Fructose 6-Phosphate-1-Kinase From Bacillus Stearothermophilus and Molecular Cloning and Sequencing of a Partial Human Muscle Fructose 6-Phosphate-1-Kinase Gene.
Date of Award
Doctor of Philosophy (PhD)
Simon H. Chang
Ezzat S. Younathan
Fructose 6-phosphate-1-kinase (PFK: EC. 126.96.36.199) catalyzes the phosphorylation of $\beta$-fructose 6-phosphate (Fru 6-P) to $\beta$-fructose 1,6-bisphosphate. The first part of this dissertation shows that PFK from Bacillus stearothermophilus (BsPFK) unlike the major PFK from E. coli (EcPFKA) exhibits a hyperbolic profile with respect to Fru 6-P concentration in the absence of phosphoenolpyruvate (PEP). BsPFK is sensitive to inhibition by high ATP concentrations and competitively inhibited by GDP or ADP. Arg-252 residue of BsPFK has been proposed to be involved in the binding of Fru 6-P. Mutation of this residue to alanine (RA252) increases BsPFK affinity for its inhibitor PEP (a 68-fold difference compared to wild type) and dramatically decreases Fru 6-P affinity (1,500-fold increase in Km). Its higher sensitivity to ATP inhibition is relieved by ADP, GDP or higher Fru 6-P concentrations. Mutation of Arg-252 to lysine (RK252) increases the affinity of the enzyme for PEP by only 2-fold and increases its Km for Fru 6-P by only 50-fold. The data indicate that the Arg-252 residue plays a major role in both Fru 6-P binding and allosteric interaction between the subunits. Arg-25, Arg-211 and Asp-59 residues have been mutated to alanine; and Asp-59 also to methionine. Arg-25 mutant has half the affinity for Fru 6-P compared to the wild type and exhibits sigmoidicity with respect to this substrate (Hill # = 2.0). Asp-59 $\to$ Ala mutant has the same affinity for PEP as the wild type whereas Asp-59 $\to$ Met mutant has 3-fold higher affinity for this modulator and the inhibition is reversed by GDP. Arg-25 and Arg-211 mutants are 100-fold less sensitive to PEP inhibition which is not relieved by GDP. The data prove unequivocally that Arg-25 and Arg-211, but not Asp-59, are involved in the direct binding of PEP and GDP. The last part of this dissertation involves isolation of a clone containing the nontranslated 5$\sp\prime$ flanking region of the human muscle PFK (HMPFK) gene. It has an additional intron extending from nucleotide $-$97 to $-$9 upstream of the ATG start codon. Furthermore, HMPFK gene is more AT rich than the rabbit gene.
Valdez, Benigno Calderon, "Site-Directed Mutagenesis in Fructose 6-Phosphate-1-Kinase From Bacillus Stearothermophilus and Molecular Cloning and Sequencing of a Partial Human Muscle Fructose 6-Phosphate-1-Kinase Gene." (1988). LSU Historical Dissertations and Theses. 4688.