Phosphorus-31 Nmr Relaxation Studies of Diethyl P- Methoxyphenyl Phosphate Bound to Phosphotriesterase

The effect of MnZ÷/Mn 2÷, Mn2÷/Zn 2÷ and ~VIn2+]Cd 2÷ reconstituted phosphotriesterase on the 3~p spin lattice (1/Tx) relaxation rate of diethyl p-methoxyphenyl phosphate has been investigated. In the presence of MnZ÷/Mn 2+ phosphotriesterase, the spin lattice relaxation rate of the phosphorus atom is enhanced giving an upper limit for the phosphorus-metal root mean-sixth average distance of 4.2 A. These results demonstrate for the first time that substrates for phosphotriesterase bind in close proximity to the binuclear metal center. Introduction Phosphotriesterase from Pseudomonas diminuta is a zinc metalloenzyme that catalyzes the hydrolysis of a broad spectrum of organophosphate tdesters including organophosphate insecticides and chemical warfare agents, t'2 Phosphotriesterase is remarkably efficient in catalyzing the hydrolysis of organophosphate triesters even though they are not natural occurring compounds. The best substrate found to date is paraoxon which has a turnover number for hydrolysis of l0 4 st. 2 The overall chemical mechanism for the hydrolysis of paraoxon is thought to proceed via the direct attack of an activated water molecule on the phosphorus center of paraoxon resulting in a net inversion of configuration. 3 It is presumed the metal center functions to activate the substrate and water molecule for hydrolytic attack but the exact role of the metal center in catalysis has yet to be defined. The metal center of phosphotriesterase is binuclear and the native zinc metals can be replaced with a variety of divalent metals yielding catalytically active enzyme. 4 EPR experiments with Mn2÷/Mn 2÷ reconstituted protein have shown the two metals are antiferromagnetically coupled by a common ligand. 5~6 Kinetic and l l3Cd NMR investigations have shown that the binuclear metal center consists of non-identical sites, M,, and M~. has been proposed to be the primary catalytic site, whereas the function of MI~ is unclear. 7'8 The crystal structure of the cadmium substituted phosphotriesterase has been determined recently. 9't° The two metal ions are 3.7 /~ apart and bridged by a solvent water molecule and a carbamate formed from Lys-169. To date, however, there has been no direct experimental evidence to indicate the substrate is bound at the active site in close proximity to either of the metal atoms. * Author to whom correspondence may be address (FAX: 409-845-9452).