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Title: The structure, activity and evolution of phosphofructokinase
Author: Hudson, P. J.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 1979
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Phosphofructokinase is an allosteric regulatory enzyme which controls the rate of glycolysis in the cell. This study has involved both the completion of the amino acid sequence of phosphofructokinase isolated from the thermophilic bacterium B. stearothermophilus and the solution of the tertiary structure to 2.4Å resolution by X-ray diffraction analysis. Tryptic peptides derived from the intact [2-¹⁴C]-carboxymethylated protein subunit were isolated and sequenced. These were compiled with some earlier sequence data to give the complete primary structure of 316 amino acid residues. The cysteine residue which is uniquely reactive to sulphydryl reagents was located at residue 70. Improvements were made to the published methods of purification of B. stearothermophilus phosphofructokinase which enabled the production of large crystals suitable for X-ray diffraction analysis. Two isomorphous heavy atom derivatives were prepared by crystal soaking techniques and a third single site derivative was prepared in solution by exploiting the reactivity of cysteine 70. These three derivatives were used to solve the three dimensional structure to 6Å and 2.4Å resolution. The enzyme exists as a stable active tetramer and is composed of subunits which each possess two structural domains. Three binding sites per subunit were located by diffusing substrates and effectors into the crystals. Two of these binding sites form the active site and the juxtaposition is consistent with a direct phosphoryl transfer between the substrates, ATP and fructose-6-phosphate, during the catalysed reaction. Surprisingly, the allosteric activator (ADP) and inhibitor (phosphoenolpyruvate) were found to compete for the same effector binding site located in a subunit interface. Both the phosphate group of the substrate (fructose-6-phosphate) in the active site and the β-phosphate group of ADP in the effector site are bound by amino acid side chains from two different but adjacent subunits. The mechanisms of cooperativity and allosteric regulation are discussed in terms of these intersubunit contacts. The entire amino acid sequence was fitted into the high resolution electron density maps. A plausible catalytic mechanism is suggested from the relative positions of the amino acid side chains found in the active site. The distribution of cysteine residues in rabbit muscle phosphofructokinase was also studied in the support of some earlier sequence evidence that this larger enzyme had not evolved by gene duplication.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available