| Abstract: | A bacterial phosphotriesterase purified to homogeneity from Flavobacterium and Pseudomonas diminuta has been found to require two divalent cations for maximum catalytic activity. The catalytic activity of phosphotriesterase is lost upon incubation with EDTA, 1,10-phenanthroline, or 8-hydroxyquinoline-5-sulfonic acid. Protection against inactivation by metal chelation is afforded by the binding of competitive inhibitors, suggesting that at least one metal is at or near the active site. Apoenzyme is prepared by incubation of the holoenzyme with the metal chelators, is fully reactivated by incubation of the apoenzyme with two equivalents of Zn[^2+], Co[^2+], Ni[^2+], Cd[^2+], or Mn[^2+]. The kinetic pK[a] of the phosphotriesterase varies, depending upon the exact metal composition, thus the metal ion is modulating the pK[a] of the active site residue, possibly involving metal-bound water molecule. 113Cd NMR spectra of the enzyme containing one or two equivalents of 113Cd show two resonances at 117 and 212 ppm downfield from Cd(ClO4)2. The resonance at 117 ppm is consistent with two nitrogens and two oxygens acting as the ligands to the metal center. The resonance at 212 ppm is consistent with the cadmium being ligated to the nitrogen atoms of three histidines and to one oxygen from either water or a carboxylate side chain. Dithiothreitol (competitive inhibitor) shifts the two resonances downfield indicating a direct coordination of sulfur to the cadmium in the phosphotriesterase-DTT complex. This observation may also suggest the presence of an open pocket at the metal centers... |
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