| Method |
Site-directed Mutagenesis—The pET32b(+) plasmid containing the gene for rePON1-G2E6 (ref 12) was used as a template for PCR amplification. Screening of rePON1 Mutants—The libraries were transformed into E. coli Origami B DE3 cells. Expression and Purification of rePON1-G2E6 Mutants—Wild type-like rePON1-G2E6 and its various mutants were expressed as fusion proteins with thioredoxin and His6 tags and purified. pH-rate Profile—kcat and Km values were determined for rePON1-G2E6 with TBBL (ref 18) at pH 5.8-9.4. Kinetic Measurements with rePON1 Mutants—The kinetic measurements were performed in buffer containing 50 mm Tris (pH 8.0) and 1 mm CaCl2, and aliphatic lactone hydrolysis was monitored as described previously (ref 6). A range of enzyme concentrations was used depending on the reactivity of the substrate and the mutant. Data Analysis—Kinetic parameters (kcat, Km, and kcat/Km) were obtained by fitting the data to the Michaelis-Menten equation (v0 = kcat[E]0[S]0/([S]0 + Km)). Rabbit PON3—Wild-type rabbit PON3 and its mutants were cloned, expressed, and purified analogously to rePON1 variants, except that the elution buffer did not contain glycerol. |