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Title: Physical studies on subunit interactions in oligomeric enzymes
Author: Leonard, K. R.
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 1970
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This thesis is concerned with the physical structure and the protein-protein interactions in the two oligomeric proteins, Phosphofructokinase (PFK) and Aldolase. PFK is known to play an important part in the regulation of glycolysis and the activity of the enzyme is modified by a variety of low molecular weight metabolites including ATP, AMP, F-6-P, FDP and citrate. Physical studies on PFK were, therefore, made with particular reference to the conformational changes which might accompany the regulation of activity. The studies on Aldolase are less extensive and represent a continuation of earlier work (K.R.Leonard, Part II thesis, 1966). In contrast to PFK, aldolase is not a regulatory protein. It is, however, composed of subunits and therefore serves as a useful comparison for PFK. Aldolase is also related to PFK in that they are both enzymes of Glycolysis, having adjacent places in the pathway, so that the product of the PFK reaction, FDP, is also the substrate of the Aldolase reaction. Aldolase was purchased from a commercial source and was found to be quite suitable for physical studies. The commercially available PFK, however, was not suitable for physical studies for a number of reasons, including the presence of a large amount of inactive protein which, was visible in the sedimentation patterns and a strong tendency to precipitate when dialysing against buffer to remove ammonium sulphate. The PFK used was therefore prepared in this laboratory by an adaptation of a published method. The sedimentation velocity experiments using schlieren optics to measure protein concentration confirmed that PFK undergoes reversible self-association above 0.5mg/ml. This aspect of the protein-protein interactions of PFK was investigated in some detail. Evidence for a reversible association was manifested by the appearance of the sedimentation pattern of the pure protein (three peaks with sapp of approx. 13 S, 18 S and 22-30 S). Further evidence was the behavior of the fastest peak which increased in sapp from 22 S at 0.5mg/ml to 30 S at 10mg/ml in direct contrast to the normal decrease in sapp with increasing concentration for a non-interacting species. This association of PFK was found to be prevented by mild alkaline pH, high ionic strength, low concentrations of SDS (detergent) and low concentrations of PCMB (thiol reagent). Addition of any of these reagents to PFK solutions resulted in a change ox the sedimentation pattern to a single boundary with sapp = 12 S (approx). This corresponds to the slowest sedimenting species in the pattern for native PFK at high concentration and is probably the unassociated 'Monomer' of the equilibrium. Measurement of sapp for PFK at the concentration level of the enzyme assay (approx. landmu;g/ml) gave a value of about 11 S for the active species, which indicated that PFK is present as the 'monomer' at this low concentration. The 12 S material, which was obtained by direct pH adjustment to pH 10.5, slowly broke down to give protein sedimenting at lower speed. The presence of 0.5mM FDP, however, had a marked stabilising effect, and protein dialysed into pH 10.5 buffer containing FDP sedimented as a sharp symmetrical boundary with s020,w of 11.9 ± 0.4. Similar concentrations of F-6-P, ATP and AMP had no stabilising effect on the 12 S protein. The diffusion coefficient of the FDP-stabilised material was measured by the spreading boundary method and a value of D020 equal to 3.2 ± 0.3 andtimes; 10-7 obtained. This combined with the sedimentation coefficient gave a value of 3.4 ± 0.3 andtimes; 105 for the molecular weight. This agreed with the value of 3.45 ± 0.1 andtimes; 105 obtained from sedimentation equilibrium. Equilibrium centrifugation studies of a series of concentrations of PFK over the range 0.5-10mg/ml enabled curves of weight-average and Z-average molecular weights against concentration for the polymerisation to be constructed. These were found to be sigmoid in shape, and the Z-average tended to a value of 3-4 andtimes; 105 at low concentration (where the 12 S species only will be present) and to a plateau of about 5.5 andtimes; (monomer mol.wt) at about 7mg/ml, followed by a slight reduction in molecular weight caused by non-ideality at higher protein concentration. These curves were compared with calculated theoretical curves for a number of model polymerising systems, and it was deduced, from the shape of the curves, that the association was to a closed polymer rather than to an indefinite, open polymerisation. A calculated curve which was a reasonable fit to the experimental points was obtained by assuming that the degree of polymerisation ,n, was six, that there was a negligible concentration of intermediate polymers present at equilibrium and that BMw (the non-ideality term) had a value close to the theoretical value for a globular protein. A tentative closed-shell structure was proposed for the hexamer. Assuming that n = 6, the equilibrium constant for the association per mole of hexamer was estimated to be 6 andtimes; 1025 Ltr5/Mole5 and hence the value of andDelta;G0 per mole of hexamer was -35 Kcal. at 20°C. Data for Mz was also obtained at 2°C and this, combined with the results at 20°C gave values of andDelta;H0 = 36 Kcal and andDelta;S0 = 240 e.u. per mole of hexamer, for the association. These results show that the principle contributing factor to the free energy of the interaction is the gain in entropy which results from the release of bound water as the monomers associate. This would implicate either hydrophobic or electrostatic interactions as chief contributors to the protein-protein binding. The effect of high salt concentration, which prevents the association, unequivocally supports the importance of electrostatic interactions in the polymerisation. PFK at pH 12 in the presence of SDS had a molecular weight of about 8.8 andtimes; 104, estimated by sedimentation equilibrium, which is about one fourth of the molecular weight obtained for the 12 S protein. The molecular weight in 6M GuHCl, 0.1M 2-mercaptoethanol was 7.6 ± 0.5 andtimes; 104 by sedimentation equilibrium and about 8 andtimes; 104 calculated from the value of the sedimentation coefficient. Allowing for experimental error, in particular in the value of the partial specific volume, these results agree with the figure obtained at high pH in the presence of SDS. Thus, the 12 S protein is made up of four subunits, each consisting of a single polypeptide chain. In order to test whether the association of PFK had any effect on the enzymic activity it was necessary to carry out the assay at the 1mg/ml enzyme level. The F-6-P activity of PFK is too high to be measured by normal spectrophotometric methods at this concentration. However, PFK will also catalyse the phosphorylation of G-l-P at a much lower rate and this activity can be assayed at the higher concentration. It was found, that there was no change in the G-l-P specific activity over the concentration range 0.2-2.0 mg/ml where the enzyme is known to associate.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available