Structure/Function Analyses of Human Serum Paraoxonase (HuPON1) Mutants Designed from a DFPase-Like Homology Model
ARMY MEDICAL RESEARCH INST OF CHEMICAL DEFENSE ABERDEEN PROVING GROUND MD
Pagination or Media Count:
Human serum paraoxonase HuPON1 is a calcium-dependent enzyme that hydrolyzes esters, including organophosphates and lactones, and exhibits anti-atherogenic properties. A few amino acids have been shown to be essential for the enzymes arylesterase and organophosphatase activities. Until very recently, a three-dimensional model was not available for HuPON1, so functional roles have not been assigned to those residues. Based on sequence-structure alignment studies, we have folded the amino acid sequence of HuPON1 onto the sixfold beta-propeller structure of squid diisopropyl-fluorophosphatase DFPase. We tested the validity of this homology model by circular dichroism CD spectroscopy and site-directed mutagenesis. Consistent with predictions from the homology model, CD data indicated that the structural composition of purified HuPON1 consists mainly of beta-sheets. Mutants of HuPON1 were assayed for enzymatic activity against phenyl acetate and paraoxon. Substitution of residues predicted to be important for substrate binding L69, H134, F222, and C284, calcium ion coordination D54, N168, N224, and D269, and catalytic mechanism of HuPON1 H285 led to enzyme inactivation. Mutants F222Y and H115W exhibited substrate-binding selectivity towards phenyl acetate and paraoxon, respectively. The homology model presented here is very similar to the recently obtained PON1 crystal structure, and has allowed identification of several residues within the enzyme active site.
- Chemical, Biological and Radiological Warfare