Evidence that several conserved histidine residues are required for hydrolytic activity of human paraoxonase/arylesterase.
Recent evidence has been acquired that implicates an important role for several histidine residues in the hydrolytic mechanisms of human paraoxonase/arylesterase (PON1). Following titration with diethylpyrocarbonate (DEPC), both human serum and recombinant human type Q PON1 were inhibited in respect to their hydrolytic activity in a dose-responsive manner. Human PON1 treated with varying concentrations lost hydrolytic activity, and with each histidine modified, there was an exponential drop in hydrolytic activity. The reaction was followed spectrophotometrically at 244 nm. Recombinant wild-type and C283A PON1 enzymes inhibited with DEPC and subsequently treated with hydroxylamine had partial restoration of activity. The C283A mutant lacks a free sulfhydryl group, indicating that its inactivation is due to histidine specific modification. The dose response and time course of inactivation as well as the extent of reactivation by hydroxylamine were similar for both the wild-type and mutant recombinant enzymes. Mutants of PON1 containing an asparagine substituted for each of several conserved histidine residues lost hydrolytic activity for each single substitution. The mutants of PON1 constructed and assayed for arylesterase activity were H114N, H133N, and H284N. Each single aminoacid substitution rendered the enzyme catalytically inactive. These two pieces of evidence implicate an important role for several histidine residues in the hydrolytic mechanism of PON1. Although it is unusual for a calcium dependent enzyme to require histidines for its catalytic activity, acquired data suggest such a circumstance.
Department of Environmental and Industrial Health, School of Public Health, University of Michigan, Ann Arbor 48109, USA.
J A
JA
Doorn
R C
RC
Sorenson
S S
SS
Billecke
C
C
Hsu
B N
BN
La Du
D000595:Amino Acid Sequence; D043303:Aryldialkylphosphatase; D002265:Carboxylic Ester Hydrolases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002478:Cells, Cultured; D017124:Conserved Sequence; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004950:Esterases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007668:Kidney / Q000166:cytology / Q000201:enzymology; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013329:Structure-Activity Relationship; D014162:Transfection
D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D006639:Histidine; D004950:Esterases; D002265:Carboxylic Ester Hydrolases; C019734:arylesterase; D043303:Aryldialkylphosphatase; C468406:PON1 protein, human; D004047:Diethyl Pyrocarbonate