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

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 (192)Q and R-192 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 R-192 PON1 and 590 for (192)Q PON1. The final purified enzymes were shown as single protein bands close to 45 kDa 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. K-m values of (192)Q and R-192 PON1 for homocysteine thiolactone were 23.5 mM and 22.6 mM respectively. For R-192 PON1, the V-max was 2.5-fold and k(cat)/K-m was 2.6-fold higher than those for (192)Q PON1 when homocysteine thiolactone is used as substrate. The present data suggest that defining (192)Q and R-192 PON1 isoforms could be a good predictor and prognostic marker in the cardiovascular risk assessment. (C) 2010 Elsevier B.V. All rights reserved.