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Alternate modes of binding in two crystal structures of alkaline phosphatase-inhibitor complexes.

ABSTRACT: Two high resolution crystal structures of Escherichia coli alkaline phosphatase (AP) in the presence of phosphonate inhibitors are reported. The phosphonate compounds, phosphonoacetic acid (PAA) and mercaptomethylphosphonic acid (MMP), bind competitively to AP with dissociation constants of 5.5 and 0.6 mM, respectively. The structures of the complexes of AP with PAA and MMP were refined at high resolution to crystallographic R-values of 19.0 and 17.5%, respectively. Refinement of the AP-inhibitor complexes was carried out using X-PLOR. The final round of refinement was done using SHELXL-97. Crystallographic analyses of the inhibitor complexes reveal different binding modes for the two phosphonate compounds. The significant difference in binding constants can be attributed to these alternative binding modes observed in the high resolution X-ray structures. The phosphinyl group of PAA coordinates to the active site zinc ions in a manner similar to the competitive inhibitor and product inorganic phosphate. In contrast, MMP binds with its phosphonate moiety directed toward solvent. Both enzyme-inhibitor complexes exhibit close contacts, one of which has the chemical and geometrical potential to be considered an unconventional hydrogen bond of the type C-H...X.

SUBMITTER: Holtz KM

PROVIDER: S-EPMC2144633 | biostudies-other | 2000 May

REPOSITORIES: biostudies-other

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Alternate modes of binding in two crystal structures of alkaline phosphatase-inhibitor complexes.

Holtz K M KM Stec B B Myers J K JK Antonelli S M SM Widlanski T S TS Kantrowitz E R ER

Protein science : a publication of the Protein Society 20000501 5

Two high resolution crystal structures of Escherichia coli alkaline phosphatase (AP) in the presence of phosphonate inhibitors are reported. The phosphonate compounds, phosphonoacetic acid (PAA) and mercaptomethylphosphonic acid (MMP), bind competitively to AP with dissociation constants of 5.5 and 0.6 mM, respectively. The structures of the complexes of AP with PAA and MMP were refined at high resolution to crystallographic R-values of 19.0 and 17.5%, respectively. Refinement of the AP-inhibito ...[more]

PMID: 10850800

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Project description:Two Zinc(ii) complexes [Zn4(L1)4]·2H2O (1) and [Zn2(L2)2]·2H2O (2) of pyruvaldehydethiosemicarbazone ligands are reported. The complexes were characterized by elemental analysis, IR, NMR, UV-vis spectroscopy and by single-crystal X-ray crystallography. X-ray crystal structure determinations of the complexes show that though Zn : ligand stoichiometry is 1 : 1 in both the complexes, the molecular unit is tetranuclear for 1 and binuclear for 2. Both the complexes show selective sensing of ATP at pH 7.4 (0.01 M HEPES) in CH3CN-H2O (9 : 1) medium in the presence of other anions like AcO-, NO3 -, F-, Cl-, H2PO4 -, HPO4 2- and P2O7 2-. The UV-titration experiments of complexes 1 and 2 with ATP results in binding constants of 2.0(±0.07) × 104 M-1 and 7.1(±0.05) × 103 M-1 respectively. The calculated detection limits of 6.7 μM and 1.7 μM for 1 and 2 respectively suggest that the complexes are sensitive detectors of ATP. High selectivity of the complexes is confirmed by the addition of ATP in presence of an excess of other anions. DFT studies confirm that the ATP complexes are more favorable than those with the other inorganic phosphate anions, in agreement with the experimental results. Phosphatase like activity of both complexes is investigated spectrophotometrically using 4-nitrophenylphosphate (NPP) as a substrate, indicating the complexes possess significant phosphate ester hydrolytic efficiency. The kinetics for the hydrolysis of the substrate NPP was studied by the initial rate method at 25 °C. Michaelis-Menten derived kinetic parameters indicate that rate of hydrolysis of the P-O bond by complex 1 is much greater than that of complex 2, the k cat values being 212(±5) and 38(±2) h-1 respectively. The DNA binding studies of the complexes were investigated using electronic absorption spectroscopy and fluorescence quenching. The absorption spectral titrations of the complexes with DNA indicate that the CT-DNA binding affinity (K b) of complex 1 (2.10(±0.07) × 106 M-1) is slightly greater than that of 2 (1.11(±0.04) × 106 M-1). From fluorescence spectra the apparent binding constant (K app) values were calculated and they are found to be 5.41(±0.01) × 105 M-1 for 1 and 3.93(±0.02) × 105 M-1 for 2. The molecular dynamics simulation demonstrates that the Zn(ii) complex 1 is a good intercalator of DNA.

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Alternate modes of binding in two crystal structures of alkaline phosphatase-inhibitor complexes.

Publications

Alternate modes of binding in two crystal structures of alkaline phosphatase-inhibitor complexes.

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