Identification of Residues Essential for Human Paraoxonase (PON1) Arylesterase/Organophosphatase Activities

Abstract

Human serum paraoxonase (PON1) is a calcium-dependent organophosphatase. To identify residues essential for PON1 activity, we adopted complementary approaches based on chemical modification and site-directed mutagenesis. To detect 45Ca2+ binding to native and chemically modified PON1, we performed nondenaturating gel electrophoresis. The environment of calcium-binding sites was probed using the Ca2+ analogue, terbium. Tb3+ binds to calcium-binding sites as shown by displacement of 45Ca2+ by Tb3+. Binding of Tb3+ is accompanied by a complete loss of enzyme activity. PON1 chemical modification with the Trp-selective reagent, N-bromosuccinimide, and the Asp/Glu-selective, dicyclohexylcarbodiimide, established that Trp and Asp/Glu residues are components of the PON1 active center and calcium-binding sites. Additional evidence for the presence of a Trp residue in the PON1 calcium-binding sites was a characteristic fluorescence emission at 545 nm from the PON1-Tb3+ complex and abolishment of that fluorescence upon modification by N-bromosuccinimide. The importance of aromatic/hydrophobic character of the residue 280 was demonstrated by site-directed mutagenesis: the W280F mutant was fully active while the W280A and W280L mutants had markedly reduced activity. Twelve amino acids among conserved His and Asp/Glu residues were found essential for PON1 arylesterase and organophosphatase activities: H114, H133, H154, H242, H284, D53, D168, D182, D268, D278, E52, and E194. Finally, the cysteines constituting the PON1 disulfide bond (C41 and C352) were essential, but the glycan chains linked to Asn 252 and 323 were not essential for PON1 secretion and activity.

Drosophila are protected from Pseudomonas aeruginosa lethality by transgenic expression of paraoxonase-1

“…While da-GAL4/+ (da, daughterless) flies (control flies) did not express PON1, UAS-PON1/da-GAL4 flies (PON1 transgenic flies) expressed PON1, with variable levels of PON1 protein expression observed (PON1R9c > PON1R31 > PON1R1 ≈ PON1R4 > PON1R19) ( Figure 3A). Multiple PON1 bands with differing electrophoretic mobility were observed and are consistent with prior reports of PON1 undergoing N-linked core glycosylation (8,31).…”

Rabbit Serum Paraoxonase 3 (PON3) Is a High Density Lipoprotein-associated Lactonase and Protects Low Density Lipoprotein against Oxidation

“…Sixteen of these residues in human PON1 were identified as essential for the hydrolytic activity, and all of them except His-243 and Trp-281 are conserved in rabbit PON3. The two PON1 mutants, W281L and H243K, corresponding to the PON3 sequence, have markedly reduced arylesterase and organophosphatase activity (32,33). This observation may at least in part explain the limited arylesterase and paraoxonase activity of rabbit serum PON3 we observed.…”

“…Although the active site of the PON1 has not been established, some residues essential for the hydrolytic 3 C. Watson, personal communication. activity have been identified by both chemical modification and site-directed mutagenesis (32)(33)(34). The chemical modification of tryptophan, histidine, aspartic acid/glutamic acid, or cysteine residues reduced or abolished enzymatic activity.…”

The Histidine 115-Histidine 134 Dyad Mediates the Lactonase Activity of Mammalian Serum Paraoxonases

“…The enzyme has two calciumbinding sites: the higher affinity calcium is required for the structural integrity, whereas the lower affinity calcium is involved in catalysis (9). Early mechanistic studies using chemical labeling and site-directed mutagenesis identified several residues that are involved in the phosphotriesterase and esterase activities of human PON1 (10,11). However, because these studies were conducted before the three-dimensional structure of PON1 was known, it was largely unclear whether these amino acids are indeed in the PON1 active site or whether they are involved in substrate binding, Ca 2ϩ binding, or catalysis.…”