EXAFS analysis of the active site of the nonmetalloenzyme human prostatic acid phosphatase by means of Cu2+ inactivation

Möller, Karin; Etten, Robert L. Van

doi:10.1002/(sici)1520-6343(1997)3:2<85::aid-bspy1>3.0.co;2-r

Cited by 3 publications

(1 citation statement)

References 28 publications

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“…In addition, X-ray absorption near edge structure (XANES) measured in the same experiment with EXAFS contains information about the binding energy and oxidation state of the selected element (34). EXAFS has been proven to work very well with solutions of metal complexes (35,36). The method has been used to study the inner shell structure of metal-HS complexes (14,(37)(38)(39)(40)(41), but its full potential and limitations in the structural studies of HS have to be explored in more detail.…”

Section: Introductionmentioning

confidence: 99%

EXAFS Study of the Inner Shell Structure in Copper(II) Complexes with Humic Substances

Korshin

Frenkel

Stern

1998

Environ. Sci. Technol.

123

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The structure of humic substances (HS) and their complexes with metal cations is a matter of ongoing debate. In this paper, the structure of Cu2+−HS complexes was studied by extended X-ray absorption fine structure spectroscopy (EXAFS). This method is highly sensitive to the local structure around the target (e.g., Cu2+ or other cations). The CuO6 octahedron was used to model the inner complexation shell of the Cu2+−HS complexes. The quality of the fit of the EXAFS data was tested using model systems (copper(II)−aqua and −salicylate complexes). On the basis of the dissimilarity of the EXAFS spectra for the Cu2+−tetrahydrofurantetracarboxylic acid complex from those of the Cu2+−HS complexes, it is concluded that complexation sites of HS are not likely to contain carboxylic groups attached to a furan ring. The Cu−O distances in all Cu2+−HS complexes were shorter than those in aqua or salicylate complexes. The contraction of the Cu−O distance was especially prominent for the axial direction, and the distortion of the inner shell was much reduced. The mean-square disorder values for the axial Cu−O pairs in Cu2+−HS complexes indicate that the axial oxygens are more tightly bound with the central cation than those in either aqua or salicylic complexes, possibly indicating six-dentate coordination of Cu2+ by HS.

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Section: Introductionmentioning

confidence: 99%

EXAFS Study of the Inner Shell Structure in Copper(II) Complexes with Humic Substances

Korshin

Frenkel

Stern

1998

Environ. Sci. Technol.

123

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Cooperative kinetics of human prostatic acid phosphatase

Luchter-Wasylewska

2001

Biochimica et Biophysica Acta (BBA) - Protein Structure and Mol

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Covalent modification and site-directed mutagenesis of an active site tryptophan of human prostatic acid phosphatase.

Zhang

Ostanin²,

Etten³

1997

Acta Biochim Pol

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Because tryptophans are found as part of the phosphate binding sites in a number of proteins, human prostatic acid phosphatase (hPAP) was examined for the presence and the role of essential tryptophan residues. The pH dependence of the intrinsic fluorescence of hPAP resembled the kinetic pH dependence. Chemical modification by N-bromosuccinimide (NBS) resulted in an inactivation of the enzyme and produced a characteristic reduction of the protein absorbance at 280 nm. Two tryptophans per subunit were modified, and this was accompanied by an apparently complete loss of enzymatic activity. In the presence of the competitive inhibitor L-(+)-tartrate, the loss of enzyme activity was significantly reduced as compared to the rate of tryptophan modification. After labeling the protein with 2,4-dinitrophenylsulfenyl chloride (DNPS-Cl), two tryptic peptides containing DNPS-labeled tryptophans were isolated and the sequences were identified by amino acid sequence analysis and mass spectroscopy. One peptide corresponded to residues 172-176, and included Trp174. The other corresponded to the C-terminal sequence, including Trp336. It was concluded that Trp174 was at the active site of the human enzyme because it was protected by the competitive inhibitor tartrate in the DNPS-Cl modification studies. This is also consistent with the location of a homologous residue in the structure of the rat enzyme. Using site-directed mutagenesis, Trp174 was replaced by Phe or Leu. Both mutants showed altered kinetic properties, including lower Km values with several aromatic substrates, and also exhibited reduced stability towards urea denaturation.

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EXAFS analysis of the active site of the nonmetalloenzyme human prostatic acid phosphatase by means of Cu2+ inactivation

Cited by 3 publications

References 28 publications

EXAFS Study of the Inner Shell Structure in Copper(II) Complexes with Humic Substances

EXAFS Study of the Inner Shell Structure in Copper(II) Complexes with Humic Substances

Cooperative kinetics of human prostatic acid phosphatase

Covalent modification and site-directed mutagenesis of an active site tryptophan of human prostatic acid phosphatase.

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