Identification of residues essential for human paraoxonase (PON1) arylesterase/organophosphatase activities

Human serum paraoxonase (PON1) is a calcium-dependent organophosphatase. To identify residues essential for PON1 activity, we adopted complementary ap proaches based on chemical modification and site-directed mutagenesis. To d etect Ca-45(2+) binding to native and chemically modified PON1, we performe d nondenaturating gel electrophoresis. The environment of calcium-binding s ites was probed using the Ca2+ analogue, terbium. Tb3+ binds to calcium-bin ding sites as shown by displacement of Ca-45(2+) by Tb3+. Binding of Tb3+ i s accompanied by a complete loss of-enzyme activity. PON1 chemical modifica tion with the Trp-selective reagent, N-bromosuccinimide, and the Asp/Glu-se lective, dicyclohexylcarbodiimide, established that Trp and Asp/Glu residue s are components of the PON1 active center and calcium-binding sites. Addit ional evidence for the presence of a Trp residue in the PON1 calcium-bindin g sites was a characteristic fluorescence emission at 545 nm from the PON1- Tb3+ complex and abolishment of that fluorescence upon modification by N-br omosuccinimide. The importance of aromatic/ hydrophobic character of the re sidue 280 was demonstrated by site-directed mutagenesis: the W280F mutant w as fully active while the W280A and W280L mutants had markedly reduced acti vity. Twelve amino acids among conserved His and Asp/Glu residues were foun d essential for PON1 arylesterase and organophosphatase activities: H114, H 133, H154, H242, H284, D53, D168, D182, D268, D278, E52, and E194. Finally, the cysteines constituting the PON1 disulfide bond (C41 and C352) were ess ential, but the glycan chains linked to Asn 252 and 323 were not essential for PON1 secretion and activity.