R. S. Bargota et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1623–1626
1625
the para position of 2a caused over 13-fold reduction in
the rate of hydrolysis (2c and 2d, respectively). Sub-
stitution of the NO2-group of paraoxon 1a with a NH2-
group rendered the substrate analogue 1b non-hydro-
lysable by serum PON1. Because compounds 1b, 1c, 5a
and 5b did not serve as substrates for serum PON1, we
tested whether these acted as inhibitors of the enzyme.21
All four derivatives caused varying degrees of inhibition
with 1b, 1c, and 5b displaying IC50 values between 1.28
and 3.27 mM (Table 1), whilst 5a was the weakest inhi-
bitor in the series (IC50 of 18.5 mM).
mode of inhibition of the paraoxonase derivatives also
needs to be evaluated. Data from these concerted efforts
will further our knowledge of the active site of PON1
and pave the way towards understanding the catalytic
mechanism of this physiologically important enzyme.
Acknowledgements
The authors thank GlaxoSmithKline for financial sup-
port, Dr. Serge Zeler for technical assistance, Dr. David
Miller for helpful discussions and critical reading of the
manuscript, and Dr. Gabra at the Birmingham Blood
Centre for donating surplus serum.
These studies provide curious observations associated
with substituent effects for carboxylic ester substrates of
PON1. Coupled with this study, we have previously
demonstrated that phenyl acetate 2a is a better substrate
than the 4-nitro analogue 2b for PON1 in that 2a shows
a 50-fold greater Vmax/Km value, despite both exhibiting
similar Km values of around 0.7 mM (unpublished
results). These findings suggest that the Lewis basicity of
the carbonyl oxygen is more important in stabilizing the
rate-limiting transition state than a low pKa for the
leaving group of structures 2a–2d. The enzyme could
stabilize the negative charge on oxygen through inter-
action with a metal ion such as Ca2+ or by protonation.
References andNotes
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The reduction in the rate of hydrolysis caused by the
presence of amine or methoxy groups on phenyl acetate
was most likely due to steric hindrance. Substrate ana-
logues of paraoxon in the phosphate ester series (1b, 1c,
5a and 5b) were all inhibitors suggesting that they bind
to PON1, but the enzyme does not catalyze their
hydrolysis at a significant rate. These inhibitors either
had an overall negative charge or were hydrophobic,
both of which characteristics were detrimental to
hydrolysis by PON1. There is a free cysteine residue at
position 283 in PON1 which is not involved in aryl-
esteraser/paraoxon catalysis. Evidence for this came
from site-directed mutagenesis (SDM) experiments in
which cysteine 283 was replaced with either alanine or
serine and the resulting mutants retained both para-
oxonase and arylesterase activities.22 Other SDM stud-
ies have revealed that several histidine and tryptophan
residues are essential for PON1 arylesterase and para-
oxonase activities.23 The aromatic nature of these amino
acids suggests that the active site of the enzyme is
hydrophobic, which could also explain why PON1
favours lipophilic substrates, such as 2a and PAF. But,
paraoxon 1a, which itself has a polar NO2 group, is also
hydrolyzed by PON1, albeit at a much reduced rate,
suggesting that there may be a different mode of inter-
action with phosphate and carboxylic acid esters.
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In summary, we have demonstrated that both electron-
withdrawing and -donating groups in the para-position
of the parent phenyl acetate esters lead to significant
reductions in the rate of hydrolysis. Using the NMR
assay described here, it should be possible to determine
Km and Vmax for these and other substrate analogues of
PON1 using purified single isomorphs. The structure–
activity data generated could then be used to extra-
polate whether the analogues have an effect on the
enzyme through binding or catalysis. In addition, the
14. (a) Antikainen, M.; Murtomaki, S.; Syvanne, M.; Pahlman,