Structural characterization of the divalent cation sites of bacterial phosphotriesterase by 113Cd NMR spectroscopy. Academic Article uri icon


  • The phosphotriesterase from Pseudomonas diminuta catalyzes the hydrolysis of organophosphate esters. The isolated native protein contains zinc, and removal of this metal abolishes the enzymatic activity. Reconstitution of the apoenzyme requires 2 mol of cadmium per mol of protein for full catalytic activity. The kcat and Km values for the hydrolysis of paraoxon for the cadmium-substituted enzyme are 4300 s-1 and 390 microM, respectively. These values compare favorably with the kinetic constants observed for the zinc-substituted enzyme (2300 s-1 and 78 microM). A hybrid enzyme containing one zinc and one cadmium ion is catalytically active, and the kinetic constants are nearly identical to the values obtained with the all-zinc-containing enzyme. The NMR spectrum of protein reconstituted with two 113Cd2+ ions per enzyme molecule exhibits cadmium resonances at 212 and 116 ppm downfield from Cd(ClO4)2. The two metal ions are, therefore, in significantly different chemical environments. These two binding sites have been designated the M alpha and M beta sites for the low- and high-field signals, respectively. Protein substituted with a single cadmium ion also shows two cadmium resonances, and thus one site is not completely filled prior to the binding of metal to the other site. The Cd/Zn hybrid protein shows a single cadmium resonance at 115 ppm, and thus the cadmium is occupying the M beta site while zinc is occupying the M alpha site. The positions of the observed chemical shifts for the two cadmium signals indicate that the ligands to both metals are composed of a mixture of oxygen and nitrogen atoms.(ABSTRACT TRUNCATED AT 250 WORDS)

published proceedings

  • Biochemistry

author list (cited authors)

  • Omburo, G. A., Mullins, L. S., & Raushel, F. M.

citation count

  • 57

complete list of authors

  • Omburo, GA||Mullins, LS||Raushel, FM

publication date

  • September 1993