1.45Å resolution crystal structure of recombinant PNP in complex with a pM multisubstrate analogue inhibitor bearing one feature of the postulated transition state

Chojnowski, Grzegorz; Breer, Katarzyna; Narczyk, Marta; Wielgus‐Kutrowska, Beata; Czapinska, H.; Hashimoto, Motomu; Hikishima, Sadao; Yokomatsu, Tsutomu; Bochtler, Matthias; Girstun, Agnieszka; Staroń, Krzysztof; Bzowska, Agnieszka

doi:10.1016/j.bbrc.2009.11.124

Cited by 8 publications

(5 citation statements)

References 25 publications

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“…There are three reasons that contribute to the stability of Pn to heterolytic cleavage arising from nucleophilic attack at the phosphorus center (Figure D): (1) a preference for a less electronegative carbon-based substituent to occupy the equatorial position of a pentacoordinate phosphoranyl intermediate (or transition state) upon attack by a nucleophile, rather than the required apical position for a leaving group (as shown in Figure D there is a 8 kcal mol –1 preference for a methoxy group to assume an apical position over a methyl group); (2) the poor leaving group ability of a carbanion relative to an alkoxide; and (3) the lack of lone pair electrons on the phosphorus bound carbon atom that prevents Lewis or general acid stabilization of a carbanion leaving group by an enzyme. For these reasons chemists have long used Pn in the design of enzyme inhibitors and mechanistic probes, − with some of these highlighted in Figure .…”

Section: Occurrence Of Organophosphonates In Naturementioning

confidence: 99%

Phosphonate Biochemistry

2016

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Organophosphonic acids are unique as natural products in terms of stability and mimicry. The C-P bond that defines these compounds resists hydrolytic cleavage, while the phosphonyl group is a versatile mimic of transition-states, intermediates, and primary metabolites. This versatility may explain why a variety of organisms have extensively explored the use organophosphonic acids as bioactive secondary metabolites. Several of these compounds, such as fosfomycin and bialaphos, figure prominently in human health and agriculture. The enzyme reactions that create these molecules are an interesting mix of chemistry that has been adopted from primary metabolism as well as those with no chemical precedent. Additionally, the phosphonate moiety represents a source of inorganic phosphate to microorganisms that live in environments that lack this nutrient; thus, unusual enzyme reactions have also evolved to cleave the C-P bond. This review is a comprehensive summary of the occurrence and function of organophosphonic acids natural products along with the mechanisms of the enzymes that synthesize and catabolize these molecules.

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Section: Occurrence Of Organophosphonates In Naturementioning

confidence: 99%

Phosphonate Biochemistry

2016

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“…The site constant coefficients will differ in cases where, for example, isomerized subunits have distinct binding attributes prior to binding (cf. half- or third-site mechanisms [8,10,11] or the conformational coupling of energetics [37]) or where the protein is heteroligomeric. The second ligand may be presented with multiple binding sites of varying affinities depending on which subunit was bound first.…”

Section: Resultsmentioning

confidence: 99%

Analytical expressions for the homotropic binding of ligand to protein dimers and trimers

Lefurgy

Leyh

2012

Analytical Biochemistry

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Cooperative binding of a ligand to multiple subsites on a protein is a common theme among enzymes and receptors. The analysis of cooperative binding data (either positive or negative) often relies on the assumption that free ligand concentration, L, can be approximated by the total ligand concentration, LT. When this approximation does not hold, such analyses result in inaccurate estimates of dissociation constants. Presented here are exact analytical expressions for equilibrium concentrations of all enzyme and ligand species (in terms of Kd values and total concentrations of protein and ligand) for homotropic dimeric and trimeric protein–ligand systems. These equations circumvent the need to approximate L and are provided in Excel worksheets suitable for simulation and least-squares fitting. The equations and worksheets are expanded to treat cases where binding signals vary with distinct site occupancy.

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“…The partial specific volume of the mutant, density, and viscosity of the buffer were calculated using the SENTERP program [49,50]. Sedimentation coefficient was calculated with HYDROPRO [51] using the structure of PNP from PDB, entry ID 3FUC [27]. This program computes the basic hydrodynamic properties, i.e., the sedimentation coefficient from the coordinates of the molecule.…”

Section: Analytical Ultracentrifugationmentioning

confidence: 99%

Trimeric Architecture Ensures the Stability and Biological Activity of the Calf Purine Nucleoside Phosphorylase: In Silico and In Vitro Studies of Monomeric and Trimeric Forms of the Enzyme

Dyzma

Wielgus‐Kutrowska

Girstun

et al. 2023

IJMS

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Mammalian purine nucleoside phosphorylase (PNP) is biologically active as a homotrimer, in which each monomer catalyzes a reaction independently of the others. To answer the question of why the native PNP forms a trimeric structure, we constructed, in silico and in vitro, the monomeric form of the enzyme. Molecular dynamics simulations showed different geometries of the active site in the non-mutated trimeric and monomeric PNP forms, which suggested that the active site in the isolated monomer could be non-functional. To confirm this hypothesis, six amino acids located at the interface of the subunits were selected and mutated to alanines to disrupt the trimer and obtain a monomer (6Ala PNP). The effects of these mutations on the enzyme structure, stability, conformational dynamics, and activity were examined. The solution experiments confirmed that the 6Ala PNP mutant occurs mainly as a monomer, with a secondary structure almost identical to the wild type, WT PNP, and importantly, it shows no enzymatic activity. Simulations confirmed that, although the secondary structure of the 6Ala monomer is similar to the WT PNP, the positions of the amino acids building the 6Ala PNP active site significantly differ. These data suggest that a trimeric structure is necessary to stabilize the geometry of the active site of this enzyme.

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1.45Å resolution crystal structure of recombinant PNP in complex with a pM multisubstrate analogue inhibitor bearing one feature of the postulated transition state

Cited by 8 publications

References 25 publications

Phosphonate Biochemistry

Phosphonate Biochemistry

Analytical expressions for the homotropic binding of ligand to protein dimers and trimers

Trimeric Architecture Ensures the Stability and Biological Activity of the Calf Purine Nucleoside Phosphorylase: In Silico and In Vitro Studies of Monomeric and Trimeric Forms of the Enzyme

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