Vanadium-51 NMR as a probe of vanadium(V) coorination to human apotransferrin

Butler, Alison; Eckert, H.

doi:10.1021/ja00190a010

Cited by 95 publications

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“…Vanadium-51 (99.75% natural abundance) NMR is a convenient method to measure the relative concentrations of vanadate species in solutions. 40 Despite the 7/2 spin, line widths for 51 V are relatively narrow and are easily resolved. 41 The 51 V NMR (Figure 2), indicated that under the conditions applied in the kinetic assays the monomer concentration maximised at ~0.6 mmol L -1 .…”

Section: Resultsmentioning

confidence: 99%

Inhibition studies of purple acid phosphatases: implications for the catalytic mechanism

Elliott¹,

Mitić²,

Gahan³

et al. 2006

J. Braz. Chem. Soc.

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Fosfatases ácidas púrpuras (PAP) pertencem à família das metalo-hidrolases binucleares que catalisam a hidrólise de um grande grupo de substratos fosfoésteres em pH ácido. Apesar dos seus sítios ativos serem estruturamente conservados as PAP apresentam versatilidade mecanística. Neste trabalho são investigados alguns aspectos do mecanismo catalítico de duas PAP, usando os inibidores vanadato e fluoreto como sondas. Enquanto as magnitudes das constantes de inibição das duas enzimas pelo vanadato são semelhantes, as enzimas diferem com relação ao modo de inibição; vanadato interage de forma não-competitiva com a PAP de porco (K i = 40 µmol L -1 ), porém inibe a PAP de feijão competitivamente (K i = 30 µmol L -1 ). De modo semelhante, o fluoreto também age como inibidor competitivo da PAP de feijão, independentemente do pH, enquanto que o fluoreto simplesmente interage com o complexo formado pela enzima de porco e seu substrato(PAPsubstrato) em pH baixo e inibe de forma não competitiva esta enzima em pH mais alto, independentemente da composição do íon metálico. Além disso, enquanto a inibição pelo fluoreto se dá através da interação lenta com a PAP de porco, ele se liga rapidamente ao sítio catalítico da enzima do feijão. Visto que se propõe que o vanadato e o fluoreto mimetizem o estado de transição e o nucleófilo, respectivamente, as diferenças observadas na cinética de inibição indicam sutis, porém distintas diferenças no mecanismo de reação destas enzimas.Purple acid phosphatases (PAPs) belong to the family of binuclear metallohydrolases and catalyse the hydrolysis of a large group of phosphoester substrates at acidic pH. Despite structural conservation in their active sites PAPs appear to display mechanistic versatility. Here, aspects of the catalytic mechanism of two PAPs are investigated using the inhibitors vanadate and fluoride as probes. While the magnitude of their vanadate inhibition constants are similar the two enzymes differ with respect to the mode of inhibition; vanadate interacts in a non-competitive fashion with pig PAP (K i = 40 μmol L -1 ) while it inhibits red kidney bean PAP competitively (K i = 30 μmol L -1 ). Similarly, fluoride also acts as a competitive inhibitor for red kidney bean PAP, independent of pH, while the inhibition of pig PAP by fluoride is uncompetitive at low pH and non-competitive at higher pH, independent of metal ion composition. Furthermore, while fluoride acts as a slow-binding inhibitor in pig PAP it binds rapidly to the catalytic site of the red kidney bean enzyme. Since vanadate and fluoride are proposed to act as transition state and nucleophile mimics, respectively, the observed differences in inhibition kinetics indicate subtle but distinct variations in the reaction mechanism of these enzymes.Keywords: purple acid phosphatase, catalytic mechanism, kinetics, enzyme inhibition IntroductionPurple acid phosphatases (PAPs) belong to the family of binuclear metallohydrolases, with members differing widely with respect to physicochemical properties, tissue localisation...

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Section: Resultsmentioning

confidence: 99%

Inhibition studies of purple acid phosphatases: implications for the catalytic mechanism

Elliott¹,

Mitić²,

Gahan³

et al. 2006

J. Braz. Chem. Soc.

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“…The interaction can readily be detected by differential UV titration [36,62], 51 V NMR [59,63,64], ultrafiltration [59,65] and calorimetry [42]; other special techniques, such as 14 N or 35 Cl NMR [19], have also been applied. The coordination takes place in the cationic form (VO 2 + ): the stability constants of the apoTf-V V interactions are 3-4 orders of magnitude higher than those with inorganic anions (phosphate, sulfate, hydrogencarbonate) [62].…”

Section: V Interactions With Apotransferrinmentioning

confidence: 99%

The speciation of vanadium in human serum

Jakusch

Pessoa²,

Kiss

2011

Coordination Chemistry Reviews

104

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“…Butler et al [17] reported a 51 V NMR spectrum of human transferrin (Tf) d = À529.5/À531.5 ppm. Rehder et al [18] measured 51 V NMR chemical shifts of bovine apo-transferrin (Tf) d = À515 ppm and of bovine prostatic acid phosphatase (Pp) d = À542 ppm in solution.…”

Section: Introductionmentioning

confidence: 99%

⁵¹V NMR Chemical Shifts Calculated from QM/MM Models of Vanadium Chloroperoxidase

Waller

Bühl

Geethalakshmi

et al. 2007

Chemistry A European J

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(51)V NMR chemical shifts calculated from QM/MM-optimized (QM=quantum mechanical; MM=molecular mechanical) models of vanadium-dependent chloroperoxidase (VCPO) are presented. An extensive number of protonation states for the vanadium cofactor (active site of the protein) and a number of probable positional isomers for each of the protonation states are considered. The size of the QM region is increased incrementally to observe the convergence behavior of the (51)V NMR chemical shifts. A total of 40 models are assessed by comparison to experimental solid-state (51)V NMR results recently reported in the literature. Isotropic chemical shifts are found to be a poor indicator of the protonation state; however, anisotropic chemical shifts and the nuclear quadrupole tensors appear to be sensitive to changes in the proton environment of the vanadium nuclei. This detailed investigation of the (51)V NMR chemical shifts computed from QM/MM models provides further evidence that the ground state is either a triply protonated (one axial water and one equatorial hydroxyl group) or a doubly protonated vanadate moiety in VCPO. Particular attention is given to the electrostatic and geometric effects of the protein environment. This is the first study to compute anisotropic NMR chemical shifts from QM/MM models of an active metalloprotein for direct comparison with solid-state MAS NMR data. This theoretical approach enhances the potential use of experimental solid-state NMR spectroscopy for the structural determination of metalloproteins.

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Vanadium-51 NMR as a probe of vanadium(V) coorination to human apotransferrin

Cited by 95 publications

References 0 publications

Inhibition studies of purple acid phosphatases: implications for the catalytic mechanism

Inhibition studies of purple acid phosphatases: implications for the catalytic mechanism

The speciation of vanadium in human serum

⁵¹V NMR Chemical Shifts Calculated from QM/MM Models of Vanadium Chloroperoxidase

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Vanadium-51 NMR as a probe of vanadium(V) coorination to human apotransferrin

Cited by 95 publications

References 0 publications

Inhibition studies of purple acid phosphatases: implications for the catalytic mechanism

Inhibition studies of purple acid phosphatases: implications for the catalytic mechanism

The speciation of vanadium in human serum

51V NMR Chemical Shifts Calculated from QM/MM Models of Vanadium Chloroperoxidase

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Product

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⁵¹V NMR Chemical Shifts Calculated from QM/MM Models of Vanadium Chloroperoxidase