Date of Award

1987

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Abstract

A system is presented for determining the relationships between structure and function in the allosteric enzyme 6-phosphofructo-1-kinase (PFK). The ATP-dependent phosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate catalyzed by PFK is the first reaction unique to the glycolytic pathway. The structure-function relationships in PFK can now be addressed by site-specific mutagenesis. This is a method for directly testing hypotheses concerning the functions of individual amino acids in a protein molecule. The main body of this dissertation is composed of three sections. The first two sections describe the development of a system by which the structure-function relationships in Bacillus stearothermophilus PFK (Bs -PFK) can be investigated using the technique of site-specific mutagenesis. The third section describes preliminary efforts made toward establishing a similar system for the PFK from rabbit muscle. (I) Nucleotide sequence of the 6-phosphofructo-1-kinase gene from Bacillus stearothermophilus and comparison with the homologous Escherichia coli gene: This section describes the cloning and sequencing of the gene encoding Bs -PFK. A significant degree of homology exists when the deduced amino acid sequence of Bs-PFK is compared with the sequences of rabbit muscle PFK or the major PFK from E. coli. (II) High-level expression of Bacillus stearothermophilus 6-phosphofructo-1-kinase in Escherichia coli: This section describes the subcloning of the Bs -PFK gene into a plasmid vector and the high level of Bs -PFK expression which results when this construction is introduced into a PFK null strain of E. coli. This high level of Bs -PFK expression completes the system required for determining the relationships between structure and function in Bs -PFK by site-specific mutagenesis. (III) Molecular cloning and sequencing of a partial cDNA for rabbit muscle 6-phosphofructo-1-kinase: The nucleotide sequence of the cDNA described in this section confirms corresponding portions of the genomic sequence for rabbit muscle PFK. The cloning of the rabbit muscle PFK cDNA fragment represents significant progress toward the long-term goal of using site-specific mutagenesis to determine the structure-function relationships in this allosteric enzyme.

Pages

118

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