Identifying how enzymes stabilize high-energy species along the reaction pathway is central to explaining their enormous rate acceleration. β-Phosphoglucomutase catalyses the isomerization of ...β-glucose-1-phosphate to β-glucose-6-phosphate and appeared to be unique in its ability to stabilize a high-energy pentacoordinate phosphorane intermediate sufficiently to be directly observable in the enzyme active site. Using 19 F-NMR and kinetic analysis, we report that the complex that forms is not the postulated high-energy reaction intermediate, but a deceptively similar transition state analogue in which MgF 3 − mimics the transferring PO 3 − moiety. Here we present a detailed characterization of the metal ion–fluoride complex bound to the enzyme active site in solution, which reveals the molecular mechanism for fluoride inhibition of β-phosphoglucomutase. This NMR methodology has a general application in identifying specific interactions between fluoride complexes and proteins and resolving structural assignments that are indistinguishable by x-ray crystallography. enzyme mechanism fluoride inhibition NMR structure phosphoryl transfer isosteric isoelectronic transition state analogue
Well‐balanced: 19F NMR spectroscopy defined a trifluoromagnesate complex for protein kinase A (multicolored) with adenosine diphosphate (black), the MgF3− ion (green), and the SP20 peptide substrate ...(purple with dots). A sphere (cyan) centered on the MgF3− ion embraces all catalytic components and much of the SP20 substrate. The content of the sphere is uncharged conforming to the charge balance hypothesis.
Previous management learning research on phronesis or practical wisdom has focussed on its local, contextual application rather than its connection to wider ethical and political relationships. ...Drawing on a new materialities perspective that is helpful in theorising the interconnectedness of contextual practical knowledge and the greater good, this article suggests phronesis is learned by understanding relationships between materialities in different spaces over time. The article is based on a qualitative, longitudinal study of Operation Princess, an actual project that took place on mainland Britain’s railway. Through a composite fiction, we show how under nationalised organisation managers learned phronesis by becoming steeped in wider material relationships over time. Privatisation introduced a financialised perspective that was detached from wider material connections and produced unworkable plans. Operation Princess was rescued by managers who had developed a phronetic appreciation of the interrelationships between employees, trains, services and communities. They learned phronesis through an institutional lengthy formative process of becoming entangled in vital materialities, repeatedly experimenting and doing, supported by intermittent classroom learning. This is a very different approach to the commodified style of management development today which abstracts and condenses, undermining the development of practical wisdom.
Human phosphoglycerate kinase (PGK) is an energy generating glycolytic enzyme that catalyses the transfer of a phosphoryl group from 1,3-bisphosphoglycerate (BPG) to ADP producing 3-phosphoglycerate ...(3PG) and ATP. PGK is composed of two α/β Rossmann-fold domains linked by a central α-helix and the active site is located in the cleft formed between the N-domain which binds BPG or 3PG, and the C-domain which binds the nucleotides ADP or ATP. Domain closure is required to bring the two substrates into close proximity for phosphoryl transfer to occur, however previous structural studies involving a range of native substrates and substrate analogues only yielded open or partly closed PGK complexes. X-ray crystallography using magnesium trifluoride (MgF
3
−
) as a isoelectronic and near-isosteric mimic of the transferring phosphoryl group (PO
3
−
), together with 3PG and ADP has been successful in trapping human PGK in a fully closed transition state analogue (TSA) complex. In this work we report the
1
H,
15
N and
13
C backbone resonance assignments of human PGK in the solution conformation of the fully closed PGK:3PG:MgF
3
:ADP TSA complex. Assignments were obtained by heteronuclear multidimensional NMR spectroscopy. In total, 97% of all backbone resonances were assigned in the complex, with 385 out of a possible 399 residues assigned in the
1
H–
15
N TROSY spectrum. Prediction of solution secondary structure from a chemical shift analysis using the TALOS-N webserver is in good agreement with the published X-ray crystal structure of this complex.
β-Phosphoglucomutase (βPGM) is a magnesium-dependent phosphoryl transfer enzyme that catalyses the reversible isomerisation of β-glucose 1-phosphate and glucose 6-phosphate, via two phosphoryl ...transfer steps and a β-glucose 1,6-bisphosphate intermediate. Substrate-free βPGM is an essential component of the catalytic cycle and an understanding of its dynamics would present significant insights into βPGM functionality, and enzyme catalysed phosphoryl transfer in general. Previously, 30 residues around the active site of substrate-free βPGM
WT
were identified as undergoing extensive millisecond dynamics and were unassignable. Here we report
1
H,
15
N and
13
C backbone resonance assignments of the P146A variant (βPGM
P146A
) in its substrate-free form, where the K145–A146 peptide bond adopts a
trans
conformation in contrast to all crystal structures of βPGM
WT
, where the K145–P146 peptide bond is
cis.
In βPGM
P146A
millisecond dynamics are suppressed for all but 17 residues, allowing 92% of backbone resonances to be assigned. Secondary structure predictions using TALOS-N reflect βPGM crystal structures, and a chemical shift comparison between substrate-free βPGM
P146A
and βPGM
WT
confirms that the solution conformations are very similar, except for the D137–A147 loop. Hence, the isomerisation state of the 145–146 peptide bond has little effect on structure but the
cis
conformation triggers millisecond dynamics in the hinge (V12–T16), the nucleophile (D8) and residues that coordinate the transferring phosphate group (D8 and S114–S116), and the D137–A147 loop (V141–A142 and K145). These millisecond dynamics occur in addition to those for residues involved in coordinating the catalytic Mg
II
ion and the L44–L53 loop responsible for substrate discrimination.
19F-based NMR analysis and hydrogen/deuterium primary isotope shifts establish the formation of a highly populated solution-state trigonal bipyramidal complex involving β-phosphoglucomutase (β-PGM), ...α-galactose 1-phosphate (αGal1P), and trifluoromagnesate (MgF3 −), PGM-MgF3-αGal1P, that is a transition state analogue for phosphoryl transfer. Full backbone resonance assignment of the protein shows that its structure is in the closed conformation required for catalytic activity and is closely related to the corresponding complex with glucose 6-phosphate, which we have recently identified using NMR analysis in solution and X-ray crystallography in the solid state. The previous identification of three structural waters in a PGM-αGal1P binary substrate complex had indicated that, in the presence of αGal1P, magnesium ions, and fluoride, β-PGM should indeed form a PGM-MgF3-αGal1P-TSA complex whereas, in the solid-state, apparently it did not. This cast doubt on the validity of the interpretation of MgF3 − complexes. The present work establishes that, in solution, the expectation that a PGM-MgF3-αGal1P-TSA complex should readily form is fulfilled. These results thus refute the final evidence used to claim that the trigonal bipyramidal species observed in some solid-state structures of complexes involving β-PGM are pentaoxyphosphorane intermediates.
The isomerization of β-glucose-1-phosphate (βG1P) to β-glucose-6-phosphate (G6P) catalyzed by β-phosphoglucomutase (βPGM) has been examined using steady- and presteady-state kinetic analysis. In the ...presence of low concentrations of β-glucose-1,6-bisphosphate (βG16BP), the reaction proceeds through a Ping Pong Bi Bi mechanism with substrate inhibition (k cat = 65 s−1, K βG1P = 15 μM, K βG16BP = 0.7 μM, K i = 122 μM). If αG16BP is used as a cofactor, more complex kinetic behavior is observed, but the nonlinear progress curves can be fit to reveal further catalytic parameters (k cat = 74 s−1, K βG1P = 15 μM, K βG16BP = 0.8 μM, K i = 122 μM, K αG16BP = 91 μM for productive binding, K αG16BP = 21 μM for unproductive binding). These data reveal that variations in the substrate structure affect transition-state affinity (approximately 140 000-fold in terms of rate acceleration) substantially more than ground-state binding (110-fold in terms of binding affinity). When fluoride and magnesium ions are present, time-dependent inhibition of the βPGM is observed. The concentration dependence of the parameters obtained from fitting these progress curves shows that a βG1P·MgF3 −·βPGM inhibitory complex is formed under the reaction conditions. The overall stability constant for this complex is approximately 2 × 10−16 M5 and suggests an affinity of the MgF3 − moiety to this transition-state analogue (TSA) of ≤70 nM. The detailed kinetic analysis shows how a special type of TSA that does not exist in solution is assembled in the active site of an enzyme. Further experiments show that under the conditions of previous structural studies, phosphorylated glucose only persists when bound to the enzyme as the TSA. The preference for TSA formation when fluoride is present, and the hydrolysis of substrates when it is not, rules out the formation of a stable pentavalent phosphorane intermediate in the active site of βPGM.
The direct observation of a transition state analogue (TSA) complex for tyrosine phosphorylation by a signaling kinase has been achieved using 19F NMR analysis of MEK6 in complex with ...tetrafluoroaluminate (AlF4 −), ADP, and p38α MAP kinase (acceptor residue: Tyr182). Solvent-induced isotope shifts and chemical shifts for the AlF4 − moiety indicate that two fluorine atoms are coordinated by the two catalytic magnesium ions of the kinase active site, while the two remaining fluorides are liganded by protein residues only. An equivalent, yet distinct, AlF4 − complex involving the alternative acceptor residue in p38α (Thr180) is only observed when the Tyr182 is mutated to phenylalanine. The formation of octahedral AlF4 − species for both acceptor residues, rather than the trigonal bipyramidal AlF3 0 previously identified in the only other metal fluoride complex with a protein kinase, shows the requirement of MEK6 for a TSA that is isoelectronic with the migrating phosphoryl group. This requirement has hitherto only been demonstrated for proteins having a single catalytic magnesium ion.
The evolutionary importance of hybridization and introgression has long been debated. Hybrids are usually rare and unfit, but even infrequent hybridization can aid adaptation by transferring ...beneficial traits between species. Here we use genomic tools to investigate introgression in Heliconius, a rapidly radiating genus of neotropical butterflies widely used in studies of ecology, behaviour, mimicry and speciation. We sequenced the genome of Heliconius melpomene and compared it with other taxa to investigate chromosomal evolution in Lepidoptera and gene flow among multiple Heliconius species and races. Among 12,669 predicted genes, biologically important expansions of families of chemosensory and Hox genes are particularly noteworthy. Chromosomal organization has remained broadly conserved since the Cretaceous period, when butterflies split from the Bombyx (silkmoth) lineage. Using genomic resequencing, we show hybrid exchange of genes between three co-mimics, Heliconius melpomene, Heliconius timareta and Heliconius elevatus, especially at two genomic regions that control mimicry pattern. We infer that closely related Heliconius species exchange protective colour-pattern genes promiscuously, implying that hybridization has an important role in adaptive radiation.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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