Physical studies on subunit interactions in oligomeric enzymes

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. lμg/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 × 10−7 obtained. This combined with the sedimentation coefficient gave a value of 3.4 ± 0.3 × 105 for the molecular weight. This agreed with the value of 3.45 ± 0.1 × 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 × 105 at low concentration (where the 12 S species only will be present) and to a plateau of about 5.5 × (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 × 1025 Ltr5/Mole5 and hence the value of ΔG0 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 ΔH0 = 36 Kcal and ΔS0 = 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 × 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 × 104 by sedimentation equilibrium and about 8 × 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. In order to test whether dissociation of PFK might occur at low concentration in the presence of inhibitors (reversible dissociation to an inactive form could form the basis of a regulatory mechanism), the sedimentation coefficient was measured under the conditions of normal activity and total inhibition by ATP. At approx. 1 μg/ml of protein, the sedimentation coefficient was found to be 11.5 ± 1.2 S in the presence of 6mM ATP (known to cause almost complete inactivation) and 10.6 ± 0.9 in the presence of 1mM FDP. It would appear, therefore, that dissociation is not a mechanism of the ATP inactivation. These results indicate that association or dissociation of PFK are not primary factors in the regulation of PFK activity.
Circular Dichroism (CD) and Optical Rotatory Dispersion (ORD) studies were carried out on PFK and Aldolase under various conditions. Both proteins showed near-UV bands, but whereas Aldolase also showed a measurable Cotton effect at 290nm in the ORD curve, PFK exhibited no detectable near-UV Cotton effect. The far-UV ORD and CD curves for native Aldolase and PFK were rather similar in size and shape.
The CD curves were converted into the corresponding ORD curves by application of the Kronig-Kramers transform using an Algol computer program to resolve the CD bands and a second program to carry out the transform. There was good agreement between the calculated and experimental ORD curves in the far-UV, which suggests that there is little contribution to the measurable far-UV ORD bands from optically active chromophores other than those that are observable in the far-UV CD curve. In the near-UV, the calculated curve for aldolase showed a sharp peak in the 290nm region, produced by the overlap of two near-UV CD bands of opposite sign, which satisfactorily explained the near-UV Cotton effect. The calculated curve for PFK in the near-UV showed a much smaller Cotton effect which explains why no anomaly was detectable in the experimental ORD curve.
CD studies were made on the aromatic model compounds, L-tryptophan, N-acetyl-L-tryptophan ethyl ester, L-tyrosine and N-acetyl-L-tyrosine ethyl ester and bands between 300nm and 190nm were obtained. The latter compound was also studied at high pH to assess the effect of ionisation of the phenolic group on the CD spectrum. The values for the molar ellipticities for the model compounds were- c at least an order of magnitude lower than the values which would be expected from the size of the near-UV bands of the proteins. It was therefore concluded that the near-UV optical activity of these chromophores must be enhanced by their presence in the tertiary structure of the protein.
The effect of pH on the dichroic bands of the two enzymes was correlated with the conformational changes which take place at pH 10–pH 11 for aldolase and PFK and at pH 4 for aldolase. (PFK is insoluble below pH 6 and could not therefore be studied). It was found that the near-UV bands of aldolase disappeared completely when the enzyme denatured at pH 4–pH 3.5 supporting the observation that the near-UV optical activity is enhanced by the three-dimensional structure of the native protein. At high pH, the near-UV band system of native aldolase was replaced by a single positive band with maximum ellipticity at about 255nm. This band was correlated with the red-shift of the tyrosine band at 225nm as the pH is increased. The tail of this red-shifted band is superimposed on the negative dichroic band from the peptide chromophore, producing a positive 'band' at 255nm.
PFK at high pH also showed this 255nm band, but in addition there was a small positive band centred on 290nm which was attributed to optically active tryptophan residues. As we have seen, the presence of near-UV optical activity implies some degree of tertiary structure, so that PFK even as high as pH 12 seems to behaving as a folded protein structure.
In the far-UV smaller changes in the 200nm–250nm negative peptide bands with increased pH were observed for PFK than for aldolase, which again suggests that the former enzyme is less denatured at high pH. An attempt was made to fit the far-UV bands with different percentages of the three types of regular secondary structure (α-helix, β-structure and random coil). It was unsuccessful for native aldolase and PFK and for PFK at pH 12, but a reasonable fit to the data was obtained for acid or alkali denatured aldolase.
Hydrogen ion binding data was obtained for PFK in 0.05M Na2SO4 which agreed reasonably well with the total of ionisable groups found in the amino acid analysis. Unlike aldolase (see below) it was possible to account for all the histidine residues by inspection of the titration curve. Spectrophotometric titrations were carried out in the presence and absence of 1mM FDP. It was found that FDP caused a shift of the titration curve to higher pH which was related to a change in pH from about 10.25 to about 11.5 for about two tyrosine residues per 100,000g of protein.
The earlier work on aldolase suggested that a number of the histidine residues might be titrating anomalously or be buried in the native protein, possibly associated with the inter-subunit interactions of the oligomer. The histidine titration region was therefore measured more carefully by estimating the ΔHapp for ionisation of the various groups from the temperature difference titration. The results indicated that few if any of the histidine residues are buried, but that a number may titrate with an abnormally low pH and be involved in the enzyme activity of the protein.
A temperature difference titration was also carried out over the acid pH range at which aldolase undergoes a co-operative dissociation and denaturation as shown by a step in the forward titration curve. The effect of temperature was to shift the step to lower pH with lower temperature (i.e. there is a stabilisation of the protein interactions on reducing the temperature). This result indicates that there must be a negative enthalpy change in going from the dissociated, denatured subunits to the native oligomer, which could implicate hydrogen bonding as a factor in the protein interactions.