Dramatic Differences in Organophosphorus Hydrolase Activity between Human and Chimeric Recombinant Mammalian Paraoxonase-1 Enzymes

Abstract

Human serum paraoxonase-1 (HuPON1) has the capacity to hydrolyze aryl esters, lactones, oxidized phospholipids, and organophosphorus (OP) compounds. HuPON1 and bacterially expressed chimeric recombinant PON1s (G2E6 and G3C9) differ by multiple amino acids, none of which are in the putative enzyme active site. To address the importance of these amino acid differences, the abilities of HuPON1, G2E6, G3C9, and several variants to hydrolyze phenyl acetate, paraoxon, and V-type OP nerve agents were examined. HuPON1 and G2E6 have a ten-fold greater catalytic efficiency toward phenyl acetate than G3C9. In contrast, bacterial PON1s are better able to promote hydrolysis of paraoxon, whereas HuPON1 is considerably better at catalyzing the hydrolysis of the nerve agents VX and VR. These studies demonstrate that mutations distant from the active site of PON1 have large and unpredictable effects on the substrate specificities and possibly the hydrolytic mechanisms of HuPON1, G2E6, and G3C9. The replacement of residue H115 in the putative active site with tryptophan (H115W) has highly disparate effects on HuPON1 and G2E6. In HuPON1, variant H115W loses the ability to hydrolyze VR but has improved activity toward paraoxon and VX. The H115W variant of G2E6 has similar paraoxonase activity to wild type G2E6, modest activity with phenyl acetate and VR, and increased VX hydrolysis. VR inhibits H115W HuPON1 competitively when paraoxon is the substrate and non-competitively when VX is the substrate. We have identified the first variant of HuPON1, H115W, that displays significantly enhanced catalytic activity against an authentic V-type nerve agent.Organophosphorus (OP) nerve agents are among the most toxic chemical substances identified (1). These compounds exert toxicity by readily binding to acetylcholinesterase (AChE) at the active site serine and inhibiting the ability of AChE to terminate cholinergic nerve transmissions (2).

Integrative analytical approach by capillary electrophoresis and kinetics under high pressure optimized for deciphering intrinsic and extrinsic cofactors that modulate activity and stability of human paraoxonase (PON1)

“…The third observation suggests reservations about the validity of hybrid mammalian rPON1s to reflect the conformational and functional behaviors of the human enzyme. A recent work showing dramatic differences in OPH activity between human PON1 and chimeric mammalian rPON1 [26], confirmed our opinion that hybrid rPON1s does not mimic perfectly human PON1 catalytic behavior [16].…”

“…Attempts at understanding its catalytic mechanisms are mostly supported by data provided by hybrid rPON1s. However, these enzymes do not mimic the true functioning of the human enzyme [26]. This argues for developing synergistic approaches capable to provide exhaustive information for char- Positions of PON1 residues involved in interaction with 2-HQ: overall top view of the 3D structure of a rPON1 showing the 3 helices forming the canopy that controls access of substrates to the active site, Ca2 the structural Ca 2+ (green sphere), Ca1 the catalytic Ca 2+ (red sphere) topped by the phosphate ion (sticks), the polymorphic R/Q192 site (dark blue sticks) and the His115-His134 dyad (cyan sticks).…”

Protective efficacy of catalytic bioscavenger, paraoxonase 1 against sarin and soman exposure in guinea pigs

“…Human recombinant PON1 expressed in Escherichia coli and Trichoplusia ni larvae also showed similar hydrolysis of substrates [11,24,25]. We have recently shown that purified human and rabbit serum and recombinant human PON1 efficiently hydrolyze nerve agents tabun, sarin and soman under in vitro conditions [26].…”

Recombinant paraoxonase 1 protects against sarin and soman toxicity following microinstillation inhalation exposure in guinea pigs

“…of Pages 6 2 M. Valiyaveettil et al / Toxicology Letters xxx (2011) xxx-xxx et al, 2005bFurlong et al, 2005;Li et al, 1995;Otto et al, 2009;Otto et al, 2010;Rochu et al, 2007;Stevens et al, 2008). Most of these studies were either performed under in vitro conditions, or in rodent models of OP toxicity (Duysen et al, 2010;Fu et al, 2005;Li et al, 1995;Wang et al, 2010).…”

“…PON1 has also been described as a potential catalytic bioscavenger by a number of groups due to its capacity to hydrolyze organophosphates (OPs) and chemical warfare nerve agents (CWNAs) (Costa et al, 2005b;Furlong et al, 2005;Otto et al, 2009;Otto et al, 2010;Stevens et al, 2008). It can hydrolyze large amounts of OPs and CWNAs compared to the stoichiometric bioscavenger human butyrylcholinesterase (BChE), which acts in a 1:1 ratio, and would thus require smaller doses and would reduce the cost of treatment (Doctor et al, 1991;Doctor and Saxena, 2005;Lenz et al, 2005;Lenz et al, 2007;Rochu et al, 2007).…”