35 research outputs found
Polarizable atomic multipole X-ray refinement: application to peptide crystals
A method to accelerate the computation of structure factors from an electron density described by anisotropic and aspherical atomic form factors via fast Fourier transformation is described for the first time
High-Resolution Analysis of Zn^2+ Coordination in the Alkaline Phosphatase Superfamily by EXAFS and X-ray Crystallography
Comparisons among evolutionarily related enzymes offer opportunities to reveal how structural differences produce different catalytic activities. Two structurally related enzymes, Escherichia coli alkaline phosphatase (AP) and
Xanthomonas axonopodis nucleotide pyrophosphatase/phosphodiesterase (NPP), have nearly identical binuclear Zn^2+ catalytic centers but show tremendous differential specificity for hydrolysis of phosphate monoesters or phosphate diesters. To determine if there are differences in Zn^2+ coordination in the two enzymes that might contribute to catalytic specificity, we analyzed both x-ray absorption spectroscopic and x-ray
crystallographic data. We report a 1.29-Ã… crystal structure of AP with bound phosphate, allowing evaluation of interactions at the AP metal site with high resolution. To make systematic comparisons between AP and NPP, we measured zinc extended x-ray absorption fine structure for AP and
NPP in the free-enzyme forms, with AMP and inorganic phosphate groundstate analogs and with vanadate transition-state analogs. These studies yielded average zinc–ligand distances in AP and NPP free-enzyme forms
and ground-state analog forms that were identical within error, suggesting little difference in metal ion coordination among these forms. Upon binding of vanadate to both enzymes, small increases in average metal–ligand distances were observed, consistent with an increased coordination number. Slightly longer increases were observed in NPP relative to AP, which could arise from subtle rearrangements of the active site or differences in the geometry of the bound vanadyl species. Overall, the
results suggest that the binuclear Zn^2+ catalytic site remains very similar between AP and NPP during the course of a reaction cycle