Exploring the aryl esterase catalysis of paraoxonase-1 through solvent kinetic isotope effects and phosphonate-based isosteric analogues of the tetrahedral reaction intermediate

Interactions of Paraoxonase-1 with Pharmacologically Relevant Carbamates

“…We thus estimate that the hydrolysis of PA is an energetically favoured process, by five-fold (=R•T•ln(660/80)) and by 16-fold (=R•T•ln(660/1)) kJ/mol relative to PTA and PNPA, respectively. This finding is in accordance with previous published results [15,18,38] and with the reaction mechanism model that proposes that the catalytic power of rePON1 can be mostly rationalised by concerted two-proton exchange referred to the histidine shuttle dyad. Considering a 20-fold higher catalytic efficiency (SA max /K m ) and an 80-fold higher catalytic efficiency (SA max /K m )/k sp of rePON1 towards PTA compared to that for PNPA, we have selected PTA as a replacement for PA in the following inhibition study on PON1 and carbamates.…”

“…The G2E6 variant of recombinant PON1 (rePON1) was used as a source of enzyme. RePON1 protein was expressed and purified in the Escherichia coli bacterial system according to the procedures reported previously [49] with minor modifications [18]. A single colony obtained after transformation with plasmid pET32b(+)-rePON1 into Origami B(DE3)pLysS cells was used to inoculate 10 mL of LB medium with 100 µg/mL ampicillin, 25 µg/mL chloramphenicol and 1 mM CaCl 2 and the culture was grown at 37 • C for 17 h. Then, 500 mL of LB medium containing 100 µg/mL ampicillin, 25 µg/mL chloramphenicol and 1 mM CaCl 2 was inoculated with 5 mL of overnight culture and grown at 37 • C to an OD600 of 0.7.…”

“…Structural and site-directed mutagenesis studies have provided us with much information on the role of individual amino acid residues in the active site and site-directed mutagenesis studies have provided us with much information on the role of individual amino acid residues in the active site of recombinant PON1 [14][15][16], but the exact description of all of the interactions that promote catalysis with this enzyme still remains elusive. However, its lactonase and arylesterase activities are proposed to be hydroxide-ion generated via general base catalysis by H115-H134 dyad [14,15], and quantitative structure-activity relationships [11] and recent exploration of PON1 catalysis through solvent kinetic isotope effects and phosphonate-based isosteric analogue of tetrahedral reaction intermediate [18] have suggested that this mechanism of the PON1-catalysed reaction pathway might be correct.…”

The Removal of Time–Concentration Data Points from Progress Curves Improves the Determination of Km: The Example of Paraoxonase 1

“…The G2E6 variant of rePON1 was used as a source of enzyme. RePON1 protein was expressed and purified in the Escherichia coli bacterial system according to the procedures reported previously [14] with minor modifications [24].…”

The Structure and Function of Paraoxonase-1 and Its Comparison to Paraoxonase-2 and -3

“…Temperature dependence studies on the binding and catalysis of the arylesterase activity of PON1 have suggested the following: (i) the rate-limiting step is the nucleophilic attack of the water molecule on the carbonyl group of PA; (ii) the induced fit of the active site does not play an important role for arylesterase activity; (iii) the calcium ion bound to the active site is necessary for stabilizing the substrates and transition states [33]. Additionally, the effects of the solvent kinetic isotope on the PA esterase activity of PON1 indicate that two protons contribute to the rate-limiting process of the reaction route, while moderate inhibition with the phenyl methylphosphonate anion, which is a stable isosteric analogue (i.e., transition state analogue) (Figure 3) that mimics the high-energy tetrahedral intermediate in the hydroxide-promoted hydrolysis pathway, reveals that the transition state suboptimally resembles the tetrahedral adduct [34]. Thus, all the mechanistic details of the reaction pathway of PON1-catalyzed ester hydrolysis remain unclear.…”