Allomorphy as a mechanism of post-translational control of enzyme activity

Abstract

Enzyme regulation is vital for metabolic adaptability in living systems. Fine control of enzyme activity is often delivered through post-translational mechanisms, such as allostery or allokairy. β-phosphoglucomutase (βPGM) from Lactococcus lactis is a phosphoryl transfer enzyme required for complete catabolism of trehalose and maltose, through the isomerisation of β-glucose 1-phosphate to glucose 6-phosphate via β-glucose 1,6-bisphosphate. Surprisingly for a gatekeeper of glycolysis, no fine control mechanism of βPGM has yet been reported. Herein, we describe allomorphy, a post-translational control mechanism of enzyme activity. In βPGM, isomerisation of the K145-P146 peptide bond results in the population of two conformers that have different activities owing to repositioning of the K145 sidechain. In vivo phosphorylating agents, such as fructose 1,6-bisphosphate, generate phosphorylated forms of both conformers, leading to a lag phase in activity until the more active phosphorylated conformer dominates. In contrast, the reaction intermediate β-glucose 1,6-bisphosphate, whose concentration depends on the β-glucose 1-phosphate concentration, couples the conformational switch and the phosphorylation step, resulting in the rapid generation of the more active phosphorylated conformer. In enabling different behaviours for different allomorphic activators, allomorphy allows an organism to maximise its responsiveness to environmental changes while minimising the diversion of valuable metabolites.

Fig. 1. βPGM catalytic cycle.

βPGM reaction scheme for the enzymatic conversion of βG1P to G6P via a βG16BP intermediate. The phosphoryl transfer reaction between phospho-enzyme (βPGM^P, phosphorylated at residue D8) and βG1P is illustrated with the transferring phosphate (blue) in the proximal site and the 1-phosphate (red) of βG1P in the distal site. The  phosphoryl transfer reaction between βPGM and βG16BP is shown with the transferring phosphate (red) in the proximal site and the 6-phosphate (blue) of βG16BP in the distal site.

Fig. 2. Effect of different phosphorylating agents on βPGM.

a, b Overlays of a section of ¹H¹⁵N-TROSY spectra highlighting the behaviour of residue A113. a βPGM_WT (black) populates conformer A and conformer B in slow exchange. βPGM_WT supplemented with F16BP (pink) populates phosphorylated conformer A (A^P) as the dominant species, phosphorylated conformer B (B^P) and a βPGM_WT:F16BP species (A:F16BP). βPGM_WT supplemented with βG16BP (green) populates an A:βG16BP complex. b βPGM_P146A (black) populates one conformer (conformer B). βPGM_P146A supplemented with F16BP (pink) populates conformer B and B^P. βPGM_P146A supplemented with βG16BP (green) populates a A^P:G6P complex and a B:βG16BP complex. Peaks indicated by grey asterisks correspond to the βPGM_WT:BeF₃ complex (grey; δ_N = 133.5 ppm; BMRB 17851), which is an analogue of A^P, and the Mg²⁺-saturated βPGM_D10N:βG16BP complex (grey; δ_N = 133.8 ppm; BMRB 27174), which is a mimic of the A:βG16BP complex, and are shown for comparison.

Fig. 3. Exchange behaviour in βPGM_WT.

Crystal structure of βPGM_WT (PDB 2WHE) showing residues of βPGM_WT undergoing conformational exchange on different timescales. Residues that populate two conformations in slow exchange are coloured in shades of blue according to chemical shift differences between conformer A and conformer B, with the intensity of colour and thickness of the backbone corresponding to larger values. Residues in conformer A and conformer B with missing backbone amide peaks in the ¹H¹⁵N-TROSY spectrum of βPGM_WT are coloured black, whereas missing backbone amide peaks in conformer B only are coloured purple. The amide ¹H¹⁵N coherences are likely broadened beyond detection owing to intermediate exchange on the millisecond timescale. The catalytic Mg²⁺ ion is indicated as a green sphere.

Fig. 4. Conformational plasticity of the active site of βPGM.

a, b Active sites of βPGM_WT (as conformer A) and βPGM_P146A superposed on the core domain. a Selected residues are shown as sticks for the crystal structures of βPGM_WT (grey carbon atoms; PDB 6YDL) and βPGM_P146A (dark green carbon atoms; PDB 6YDK). In βPGM_WT, a cis K145–P146 peptide bond allows coordination of the K145 sidechain by E169 and A113, whereas in βPGM_P146A a trans K146-A146 peptide bond changes significantly the backbone conformation of the D137–A147 loop, which precludes active site engagement of the K145 sidechain. The catalytic Mg²⁺ ion is drawn as a green sphere, black dashes indicate metal ion coordination and orange dashes show probable hydrogen bonds. b Selected residues, the MgF₃^– moiety and G6P are shown as sticks for the crystal structures of the βPGM_WT:MgF₃:G6P TSA complex (grey carbon atoms; PDB 2WF5) and the βPGM_P146A:MgF₃:G6P TSA complex (dark green carbon atoms; PDB 6YDJ). βPGM_WT maintains the cis K145–P146 peptide bond, whereas βPGM_P146A changes the isomerisation state of the K145–A146 peptide bond from a trans conformation in the substrate-free enzyme to a cis conformation in the transition state. c, d Omit maps generated by refinement in the absence of residues S144–P148 in βPGM_P146A. c The S144–P148 segment, containing a trans K145–A146 peptide bond, with positive difference density (Fo–Fc; green mesh contoured at +2.5σ) in substrate-free βPGM_P146A. d The S144–P148 segment, containing a cis K145–A146 peptide bond, with positive difference density (Fo–Fc; green mesh contoured at +2.5σ) in the βPGM_P146A:MgF₃:G6P TSA complex.

Fig. 5. Kinetic profiles of βPGM activity.

a, b Reaction kinetics for the conversion of βG1P to G6P catalysed by βPGM_WT and βPGM_P146A. The rate of G6P production was measured indirectly using a glucose 6-phosphate dehydrogenase coupled assay, in which G6P is oxidised and concomitant NAD⁺ reduction is monitored by the increase in absorbance at 340 nm. Reaction catalysed by either a βPGM_WT or b βPGM_P146A in standard kinetic buffer using either F16BP (circles), AcP (squares) or βG16BP (diamonds) as a phosphorylating agent. For clarity, between 100 and 8% of the data points are included in the kinetic profiles. Following βG1P substrate depletion, the kinetic profiles show an apparent increase in G6P concentration, which results from: (1) the concentration of the reaction ingredients through evaporation from the assay plate wells and (2) for the reactions recorded using βG16BP, the enzyme-dependent conversion of remaining βG16BP to G6P via βPGM^P, occurring at a rate proportional to the amount of enzyme.

Fig. 6. Kinetic model of βPGM activity.

a, b Reaction schemes for βPGM_WT as conformer A or conformer B with different phosphorylating agents. The favoured pathways are shown (red text) for βPGM_WT with a F16BP as a phosphorylating agent and b βG16BP as a phosphorylating agent. The βG16BP generating steps are highlighted in blue text. Fructose monophosphate (FMP) is either fructose 6-phosphate or fructose 1-phosphate. The complexes X:P1G6P (X = A or B) and A:P6G1P denote explicitly the orientation of βG16BP bound in the active site. The double-headed arrows connecting A^P and B^P indicate that these species interconvert with a multi-second exchange rate, similar to that described for the interconversion of conformer A and conformer B.

Fig. 7. Mechanisms of regulation and activity profiles in monomeric enzymes.

In allostery, binding (or reaction) of an allosteric effector (purple rectangle) outside of the active site shifts the enzyme population from an inactive form (red circle and red profile) to an active form (green square and green profile), which stimulates the transformation of substrate (blue oval) to product (yellow triangle) at the catalytic rate (k_cat, green arrow). In allokairy, binding of substrate in the active site shifts the enzyme population from an inactive form to an active form, at an exchange rate (k_ex) that is similar to k_cat, resulting in time-dependent activity profiles (gradient of light green to dark green profiles). Following exhaustion of substrate, the enzyme population returns to the original equilibrium position. In allomorphy, reaction of the activating substrate, termed here allomorphic full activator (green hexagon), in the active site shifts the enzyme population from an inactive form to an active form, which stimulates the transformation of the native substrate (blue oval) to product (yellow triangle) at the maximal catalytic rate (k_cat, green arrow and green profile). However, reaction of alternatives substrates, termed here allomorphic partial activators (pink pentagon), in the active site are unable to shift the enzyme population from an inactive form to an active form, resulting in a slower overall catalytic rate (k’_cat, pink arrow and pink profile). The exchange rate (k_ex) between the two enzyme forms is much slower than k_cat. Following exhaustion of the allomorphic activator, the enzyme population returns to the original equilibrium position.