Mechanism for the hydrolysis of organophosphates and investigations into the stereoselective hydrolysis of organophosphorus Esters by Phosphotriesterase.
Aubert, Sarah Dwyer
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Phosphotriesterase (PTE) is a zinc metalloenzyme that catalyzes the hydrolysis of organophosphorus compounds. Metal ion roles during binding and catalysis are probed by comparing the kinetic properties of Zn/Zn, Cd/Cd, and Zn/Cd PTE with a variety of phosphate trisesters. The metal in the α-site of the binuclear metal center modulates the pKa values determined from pH-rate profiles. These results suggest that the α-metal is responsible for activating the nucleophilic hydroxide. In an effort to determine the function of the β-metal, the kinetic parameters for diethyl p-chlorophenyl thiophosphate are compared with diethyl p-chlorophenyl phosphate. The thiophosphate substrate is hydrolyzed 20 to 100-times faster than the phosphate substrate for Zn/Zn, Cd/Cd, and Zn/Cd PTE. When Cd2+ occupies the β-site, the inverse thio effect increases which suggests polarization by the β-metal on the phosphoryl oxygen or sulfur bond. The catalytic roles of Asp 233, His 254, and Asp 301 are determined by comparing the kinetic parameters of a series of alanine and asparagine mutations with paraoxon and diethyl p-chlorophenyl phosphate. The increased rate of hydrolysis for diethyl p-chlorophenyl phosphate with the mutants is consistent with the existence of a proton relay system from Asp 301 to His 254 to Asp 233. A detailed mechanism for the hydrolysis of organophosphates by PTE has been proposed. PTE hydrolyzes a number of chiral organophosphorus esters. The pKa of the leaving group phenol is altered for a series of chiral phosphate, phosphonate, and phosphinate esters. The stereoselectivity of wild-type, G60A, and I106G/F132G/H257Y PTE is enhanced as the pKa value of the leaving group phenol increases for phosphate, phosphonate, and phosphinate esters. In addition to improving the stereoselectivity of phosphotriesterase, mutations that affect the size of the active site of PTE are screened to identify a mutant enzyme that preferentially hydrolyzes the opposite isomer of wild-type PTE. The rate constants and stereoselectivity ratios for a number of active site mutants have been determined. H254Y/L303T PTE reverses the stereoselective preference of phosphonate and phosphinate substrates. The PTE stereoselectivity of O-methyl, O-phenyl acetylphenyl phosphate is reversed 970-fold by I106G/F132G/H257Y. A reversal mutant was resolved for phosphate, phosphonate, and phosphinate esters.