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 sev eral histidine residues in the hydrolytic mechanisms of human paraoxonase/a rylesterase (PON1). Following titration with diethylpyrocarbonate (DEPC), b oth human serum and recombinant human type Q PON1 were inhibited in respect to their hydrolytic activity in a dose-responsive manner. Human PON1 treat ed with varying concentrations lost hydrolytic activity, and with each hist idine modified, there was an exponential drop in hydrolytic activity. The r eaction was followed spectrophotometrically at 244 nm. Recombinant wild-typ e 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 histidin e specific modification. The dose response and time course of inactivation as well as the extent of reactivation by hydroxylamine were similar for bot h 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 H2 84N. Each single aminoacid substitution rendered the enzyme catalytically i nactive. These two pieces of evidence implicate an important role for sever al histidine residues in the hydrolytic mechanism of PON1. Although it is u nusual for a calcium dependent enzyme to require histidines for its catalyt ic activity, acquired data suggest such a circumstance. (C) 1999 Elsevier S cience Ireland Ltd. All rights reserved.