Dramatic differences in organophosphorus hydrolase activity between human and chimeric recombinant mammalian paraoxonase-1 enzymes.
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 10-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 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 paraoxonase activity similar to that of wild-type G2E6, modest activity with phenyl acetate and VR, and enhanced VX hydrolysis. VR inhibits H115W HuPON1 competitively when paraoxon is the substrate and noncompetitively 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.
Otto
Tamara C
TC
Physiology and Immunology Branch, Research Division, U.S. Army Medical Research Institute of Chemical Defense, 3100 Ricketts Point Road, Aberdeen Proving Ground, Maryland 21010-5400, USA.
Harsch
Christina K
CK
Yeung
David T
DT
Magliery
Thomas J
TJ
Cerasoli
Douglas M
DM
Lenz
David E
DE
D043303:Aryldialkylphosphatase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001419:Bacteria / Q000235:genetics / Q000378:metabolism; D002265:Carboxylic Ester Hydrolases / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D006801:Humans; D008954:Models, Biological; D009154:Mutation; D010261:Paraoxon / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity / Q000235:genetics
D011994:Recombinant Proteins; D002265:Carboxylic Ester Hydrolases; C019734:arylesterase; D043303:Aryldialkylphosphatase; C468406:PON1 protein, human; D010261:Paraoxon