Differential hydrolysis of homocysteine thiolactone by purified human serum (192)Q and (192)R 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 (192)R 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 (192)R PON1 and 590 for (192)Q 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. K(m) values of (192)Q and (192)R PON1 for homocysteine thiolactone were 23.5mM and 22.6mM respectively. For (192)R 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 (192)R PON1 isoforms could be a good predictor and prognostic marker in the cardiovascular risk assessment.
Department of Biochemistry, Hacettepe University Faculty of Medicine, 06100, Ankara, Turkey. ahmetb@hacettepe.edu.tr
Ahmet
A
Bayrak
Tülin
T
Bayrak
Ediz
E
Demirpençe
Kamer
K
Kılınç
D000645:Ammonium Sulfate / Q000737:chemistry; D043303:Aryldialkylphosphatase / Q000097:blood / Q000737:chemistry*; D050197:Atherosclerosis / Q000378:metabolism; D015153:Blotting, Western; D011232:Chemical Precipitation; D002853:Chromatography, Liquid; D006710:Homocysteine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000097:blood / Q000737:chemistry; D007700:Kinetics; D010261:Paraoxon / Q000737:chemistry; D010648:Phenylacetates / Q000737:chemistry
D007527:Isoenzymes; D010648:Phenylacetates; D006710:Homocysteine; C007957:homocysteine thiolactone; D043303:Aryldialkylphosphatase; C468406:PON1 protein, human; D010261:Paraoxon; D000645:Ammonium Sulfate