Differential hydrolysis of homocysteine thiolactone by purified human serum 192Q and 192R PON1 isoenzymes

Abstract

Human serum paraoxonase 1 (PON1) is a HDL-associated enzyme that catalyzes the hydrolysis of a variety of aromatic carboxylic acid esters and several organophosphates. Recently it has been suggested that a physiological substrate of serum PON1 is homocysteine thiolactone which is a putative risk factor in atherosclerosis. In this study, human 192Q and 192R PON1 isoenzymes were purified from the respective phenotype human serum, using a protocol consisting of ammonium sulfate precipitation and four chromatography steps: gel filtration, ion-exchange, non-specific affinity, and a second ion-exchange. Using paraoxon as substrate, overall purification fold was found as 742 for 192R PON1 and 590 for 192Q PON1. The final purified enzymes were shown as single protein bands close to 45kDa on SDS-PAGE and confirmed by Western blot. Substrate kinetics were studied with phenyl acetate, paraoxon and homocysteine thiolactone. Both PON1 isoenzymes showed mixed type inhibition with phenyl acetate. Km values of 192Q and 192R PON1 for homocysteine thiolactone were 23.5mM and 22.6mM respectively. For 192R PON1, the Vmax was 2.5-fold and kcat/Km was 2.6-fold higher than those for 192Q PON1 when homocysteine thiolactone is used as substrate. The present data suggest that defining 192Q and 192R PON1 isoforms could be a good predictor and prognostic marker in the cardiovascular risk assessment.