Catalytic Characteristics Of Methyl Parathion Hydrolase From Plesiomonas Sp.M6

Methyl parathion hydrolase (MPH) was a novel member of phosphotriesterase. In this study, the mpd gene was subcloned and MPH was expressed using its native promoter in Escherichia coli DH5α. The protein was purified to homogeneity by a combination of DEAE-Sephadex, Bio-Gel P30 and CM-Sepharose chromatography steps. The kinetic rate constants, k_cat and k_cat/K_m, for the hydrolysis of methyl parathion are 175 s^-1 and 9.8×10⁴ M^-1 s^-1, respectively. Results showed that metal chelating compounds cannot inhabit the enzyme activity. Inductively Coupled Plasma-Atomic Emission Spectrometry analysis revealed that MPH is a zinc-containing enzyme, the Zinc to enzyme molar ratio is near 2:1. In order to investigate critical residues related to enzymatic activity of MPH, chemical modification reagents EDC, DEPC, butanedione and pyridoxal were tested. The enzyme is not inactivated by chemical modification reagents carbodimide, pyridoxal, butanedione and thus asperate, glutamate, lysine and arginine do not appear to be critical for the catalytic activity. However, the MPH is reversibly inactivated by diethyl pyrocarbonate (DEPC) and the inactivation rate is 9.6 h^-1. When adding neutralized hydroxylamine, the enzymatic activity of inactivated-MPH was restored to about 60% of initial activity. These results showed that histidine residue is essential for enzyme activity, which also can be proved by a large increase in absorbance at 245nm after the modification of histidine residues by DEPC. Unlike the catalytic mechanism of Organophosphorus hydrolase (OPH), the pH-rate profiles for V_max of the wild-type and mutant MPH using methyl parathion as a substrate indicated a requirement of two ionizable groups for full catalytic activity. Site-directed mutagenesis of eight histidine residues to asparagine showed that at least five histidine residues are involved in the catalytic activity. These substitutions resulted in dramatically change of pK_a values of some mutant enzymes. We also obtained two mutants S301A and D274V, whose kinetic constants reflected that Ser301 and Asp274 are important residues for enzyme activity. Additionally, we have screened MPH variants with altered substrate specificity using triazophos as the substrate. We got five variants, of which M5 exhibited a 85-fold increase in k_cat and 200-fold increase in k_cat/K_m values. All these results suggested that...