Transition state analogue (TSA) complexes formed by phosphoglycerate kinase (PGK) have been used to test the hypothesis that balancing of charge within transition dominates enzyme-catalyzed phosphoryl transfer. High-resolution structures trifluoromagnesate (MgF3−) and tetrafluoroaluminate (AlF4−) PGK determined using X-ray crystallography 19F-based NMR methods, revealing nature catalytically relevant this archetypal metabolic kinase. Importantly, side chain K219, which coordinates...

Phosphoglycerate kinase (PGK) is the enzyme responsible for first ATP-generating step of glycolysis and has been implicated extensively in oncogenesis its development. Solution small angle x-ray scattering (SAXS) data, combination with crystal structures complex substrate product analogues, reveal a new conformation resting state demonstrate role binding preparation domain closure. Comparison curves different states allowed complete reaction cycle to be resolved both structurally temporally....

The signal to move or proliferate diverges at RAF1.

The enzyme family harboring the post-translationally formed 5-methylene-3,5-dihydro-4H-imidazol-4-one (MIO) catalytic residue comprises both aromatic amino acid ammonia-lyases (ALs) and 2,3-aminomutases (AMs). structural origin of different functions role inner loop region in substrate binding are not fully understood. Here, we provide three-dimensional structures for Petroselinum crispum phenylalanine AL (PcPAL) with resolved loops a catalytically competent conformation. Using molecular...

The sequence of a phenylalanine ammonia-lyase (PAL; EC: 4.3.1.24) the thermophilic and radiotolerant bacterium Rubrobacter xylanophilus (RxPAL) was identified by screening genomes bacteria for members family. A synthetic gene encoding RxPAL protein cloned overexpressed in Escherichia coli TOP 10 soluble form with an N-terminal His6-tag recombinant purified Ni-NTA affinity chromatography. activity assay l-phenylalanine at various pH values exhibited local maximum 8.5 global 11.5. Circular...

The complexes of pig muscle 3-phosphoglycerate kinase with the substrate MgATP and nonsubstrate Mg2+-free ATP have been characterized by binding, kinetic, crystallographic studies. Comparative experiments ADP MgADP also carried out. In contrast to less specific largely ionic binding ADP, occupation adenosine pocket has revealed displacement anions, as well supported isothermal calorimetric titrations. nucleotides similarly stabilize overall protein structure restrict conformational...

Non-natural l -nucleoside analogues are increasingly used as therapeutic agents to treat cancer and viral infections. To be active, -nucleosides need phosphorylated their respective triphosphate metabolites. This stepwise phosphorylation relies on human enzymes capable of processing enantiomers. We crystallographic analysis reveal the molecular basis for low enantioselectivity broad specificity 3-phosphoglycerate kinase (hPGK), an enzyme responsible last step many nucleotide derivatives....

Coupling of structural flexibility and biological function is an essential feature proteins. The role relative domain movements in enzyme has been evidenced many cases. However, the way communication between protein domains its manifestation their as well are rarely delineated. In this review we summarize comprehensive studies with a typical hinge-bending two-domain enzyme, 3-phosphoglycerate kinase. A possible mechanism proposed by which two substrates that bind to different trigger...

Abstract Glycerol diglycidyl ether (GDE) is a convenient and inexpensive bisepoxide cross‐linker as demonstrated by the preparation of cross‐linked enzyme aggregates (CLEAs) from two classes. The GDE CLEAs lipase Pseudomonas fluorescens (AK), Burkholderia cepacia (PS), B Candida antarctica (CaL B) well phenylalanine ammonia‐lyase (PAL) Petroselinum crispum improved properties compared with their glutaraldehyde (GA) counterparts. Ultrasonication studies indicated that PS PAL were mechanically...

A novel phenylalanine ammonia-lyase of the psychrophilic yeast Pseudozyma antarctica (PzaPAL) was identified by screening microbial genomes against known PAL sequences. PzaPAL has a significantly different substrate binding pocket with an extended loop (26 aa long) connected to aromatic ring region active site as compared PALs from eukaryotes. The general properties recombinant expressed in E. coli were characterized including kinetic features this l-phenylalanine (S)-1a and further racemic...

RAF inhibitors achieve unprecedented but mainly transient clinical responses in patients with melanoma whose tumors harbor an activating BRAF mutation. One notable side-effect of is the stimulation cutaneous skin tumors, arising about 30% receiving these drugs, which are thought to develop as a result inhibitor-induced activation wild-type Raf occult precursor lesions. This effect raises possibility that less manageable might also arise other epithelial tissues. Here we provide preclinical...

Closure of the two domains 3‐phosphoglycerate kinase, upon substrate binding, is essential for enzyme function. The available crystal structures cannot provide sufficient information about mechanism assisted domain closure and requirement only one or both substrates, since lattice forces may hinder large scale movements. In this study known X‐ray data, obtained open closed conformations, were probed by solution small‐angle scattering experiments. results prove that binding substrates...

3‐Phosphoglycerate kinase (PGK) is a typical two‐domain hinge‐bending enzyme with well‐structured interdomain region. The mechanism of domain–domain interaction and its regulation by substrate binding not yet fully understood. Here the existence strong cooperativity between two domains was demonstrated following heat transitions pig muscle yeast PGKs using differential scanning microcalorimetry fluorimetry. Two mutants PGK containing single tryptophan fluorophore either in N‐ or C‐terminal...

3-Phosphoglycerate kinase is a hinge-bending enzyme with substrate-assisted domain closure. However, the closure mechanism has not been described in terms of structural details. Here we present experimental evidence participation individual substrate binding side chains operation main hinge which distant from sites. The combined mutational, kinetic, and (DSC SAXS) data for human 3-phosphoglycerate have shown that catalytic residue R38, also binds 3-phosphoglycerate, essential inducing...

This article overviews the numerous immobilization methods available for various biocatalysts such as whole-cells, cell fragments, lysates or enzymes which do not require preliminary enzyme purification and introduces an advanced approach avoiding costly time consuming downstream processes required by of purified enzyme-based (such chromatographic dialysis). Our is based on silica shell coated magnetic nanoparticles solid carriers decorated with mixed functions having either coordinative...

3-Phosphoglycerate kinase (PGK) is a two-domain hinge-bending enzyme. It still unclear how the geometry of active site formed during domain closure and catalytic residues are brought into optimal position for reaction. Comparison three-dimensional structures in various open closed conformations suggests large (10 Å) movement Lys 215 closure. This change would be required direct participation this side chain both catalyzed phospho transfer special anion-caused activation. To test multiple...

The energetic changes accompanying domain closure of 3‐phosphoglycerate kinase, a typical hinge‐bending enzyme, were assessed. Calorimetric titrations the enzyme with each substrate, both in absence and presence other one, provide information not only about energetics substrate binding, but associated conformational changes, including closure. Our results suggest that rearrangements hinge generated by binding substrates main driving force for overcoming slightly unfavourable contact...

Abstract A number of class I lyase‐like enzymes, including aromatic ammonia‐lyases and 2,3‐aminomutases, contain the electrophilic 3,5‐dihydro‐5‐methylidene‐4 H ‐imidazol‐4‐one (MIO) catalytic moiety. This study reveals that Pseudomonas fluorescens R124 strain isolated from a nutrient‐limited cave encodes histidine ammonia‐lyase, tyrosine/phenylalanine/histidine ammonia‐lyase (XAL), phenylalanine 2,3‐aminomutase (PAM), demonstrates an organism under nitrogen‐limited conditions can develop...

The endothelium functions as a semipermeable barrier regulating fluid homeostasis, nutrient, and gas supply to the tissue. Endothelial permeability is increased in several pathological conditions including inflammation tumors; despite its clinical relevance, however, there are no specific therapies preventing vascular leakage. Here, we show that endothelial cell‐restricted ablation of BRAF , kinase frequently activated cancer, prevents leaking well metastatic spread. regulates by promoting...

Abstract The dynamics of the actin cytoskeleton and its connection to endothelial cell–cell junctions determine barrier function cells. proper regulation opening/closing is necessary for normal vessels, dysregulation can result in chronic acute inflammation leading edema formation. By using atomic force microscopy, we show here that thrombin‐induced permeability human umbilical vein cells, associated with stress fiber formation, stiffens cell center. depletion MEK/ERK kinase BRAF reduces...

The wide specificity of 3-phosphoglycerate kinase (PGK) towards its nucleotide substrate is a property that allows contribution this enzyme to the effective phosphorylation (i.e.activation) nucleotide-based pro-drugs against HIV. Here, structural basis nucleotide-PGK interaction characterised in comparison other kinases, namely pyruvate (PK) and creatine (CK), by kinetic analysis modelling (docking) studies. results provided evidence for favouring purinevs.pyrimidine base containing...

Mechanotransduction, the process of how cells sense and convert mechanical stimuli into biochemical response, is crucial in migration leukocytes or cancer through endothelium during inflammation metastasis. Migrating exert forces on cell surface adhesion molecules, such as platelet endothelial molecule PECAM-1, this essential for a successful transmigration. To study PECAM-1-mediated mechanotransduction, we applied PECAM-1-antibody-coated magnetic beads exerted about 40 pN force monolayer....