STRUCTURAL CHARACTERIZATION OF THE DIVALENT-CATION SITES OF BACTERIAL PHOSPHOTRIESTERASE BY CD-113 NMR-SPECTROSCOPY

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 k(cat) and K(m) values for the hydrolysis of paraoxon for the cadmium-substituted enzyme are 4300 s-1 and 390 muM, respectively. These values compare favorably with the kinetic constants observed for the zinc-substituted enzyme (2300 s-1 and 78 muM). 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 Cd-113(2+) 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. Ligation by sulfur and a ligand set composed exclusively of oxygen are excluded. On the basis of observed chemical shifts of Cd-113(2+) bound to metalloenzymes of known structure it is likely that the metal bound at the M(alpha) site is ligated by three nitrogens and one oxygen, while the M(beta) site is ligated by two nitrogens and two oxygen atoms. The binding of dithiothreitol shifts each of the two cadmium resonances downfield by 100-150 ppm, and thus both metal sites are accessible to external ligands.