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.

Enzymatic production of β-glucose 1,6-bisphosphate through manipulation of catalytic magnesium coordination

“…3D). As βG16BP is the only phosphorylating agent known to induce linear initial kinetics in βPGM, 23 this experiment provided a clear demonstration of the activity of the final βG16BP product.…”

“…The slow-exchange behaviour that arises in βPGM WT from cis-trans proline isomerisation at the K145-P146 peptide bond is also observable in βPGM D170N . 23 Notably, around 15 peaks are present for βPGM D170N that are absent in the spectrum of βPGM WT . These additional peaks indicate that a backbone conformational exchange process, occurring on the millisecond timescale in βPGM WT , has been abolished in βPGM D170N .…”

“…Thus, the tight binding and high reactivity of βG16BP maintain a low steady state concentration, which precludes the harvesting of this species in useful quantities. The crystal structures of substratefree βPGM WT (PDB: 6YDL 23 ) and of the βPGM WT P analogue complex (βPGM WT :BeF 3 complex, PDB: 2WFA 15 ) indicate that the catalytic magnesium ion (Mg cat ) is coordinated through three enzyme atoms in the former and four phospho-enzyme atoms in the latter (Fig. 2).…”

“…These additional peaks indicate that a backbone conformational exchange process, occurring on the millisecond timescale in βPGM WT , has been abolished in βPGM D170N . 23 To assess the stability of βPGM D170N and to check for timedependent reversion to βPGM WT by deamidation, 25 a sample of βPGM D170N was incubated at 25°C for 48 h and both 1 H 15 N-TROSY NMR spectra and 31 P NMR time-course experiments were acquired every 24 h. A comparison of 1 H 15 N-TROSY spectra recorded for βPGM D170N preincubated at 25°C for 0 h and 48 h shows a near-identical correspondence of peaks indicating that the incubation process has a negligible effect on the integrity of βPGM D170N (Fig. S4A †).…”

An Enzyme with High Catalytic Proficiency Utilizes Distal Site Substrate Binding Energy to Stabilize the Closed State but at the Expense of Substrate Inhibition

“… 27 − 38 Substrate-free βPGM adopts an open conformation where the active site cleft, located between the cap and core domains, is exposed to bulk solvent ( Figure 1 B). 28 , 31 , 35 A cap domain rotation of 33–36° at the interdomain hinge leads to a closed transition state conformation, 31 as revealed in transition state analogue (TSA) complexes between βPGM, metallofluoride moieties, and G6P or βG1P analogues. 35 , 37 , 38 The βPGM:AlF 4 :G6P, βPGM:MgF 3 :G6P, βPGM:AlF 4 :βG1fluorophosphonate, and βPGM:MgF 3 :βG1fluorophosphonate TSA complexes mimic the active site organization for the phosphoryl transfer chemical step.…”