| abstract |
Enzymes are used for a number of industrial and medicinal reasons. To study enzymes,
small model systems are frequently used to simplify the catalytic systems that enzymes use. There
has been studies done metal ions and phosphodiesters, Ca2+ ions on phosphodiesters, metal ions
and esters. But, one particular field of model systems not studied much is the effect of Ca2+ ions
on esters. This would give us a broader understanding in which Ca2+ ions are able to interact with
carbonyl oxygens in models, even if they do not exhibit an negative charge. In order to test the
importance of the negative charge on the non-bridging oxygens in phosphodiesters for metal ion
catalysis, the effect on hydrolysis rates with Ca2+ was examined for p-nitrophenyl acetate (pNPA).
This was tested by running kinetic assays by measuring the appearance of the product, p-nitrophenolate,
using UV-Vis spectroscopy and also varying both the [-OH] and [Ca2+]. Through
these assays, it was found that Ca2+ did indeed catalyze the hydrolysis from pNPA
to p-nitrophenolate, and only one Ca2+ was involved in the catalysis. The rate constant with respect to
Ca2+ was found to be 1.51 M/M2s-1, and that using a [Ca2+]=330 mM increases the rate of
hydrolysis by approximately 77% compared to no Ca2+ ions, 1131 m/Ms-1 and 638 m/Ms-1
respectively. These data indicate that although there is an increase in hydrolysis rates for pNPA
when Ca2+ ions are present, it is very small compared to phosphodiesters, whose catalysis can be
over 800 fold. This could be due to several different factors, including the negative charge on the
phosphodiesters having a larger interaction with Ca2+ than a carbonyl oxygen Ca2+.
In order to test how Ca2+ ions catalyze the hydrolysis of pNPA specifically, the molecule
p-nitroacetanilide (pNAA) was examined. Where pNPA's hydrolysis occurs via a concerted
fashion, pNAA's occurs via a stepwise reaction. This would allow us to see if Ca2+ interacts with
the carbonyl oxygen or only the nucleophile. pNAA's tetrahedral intermediate contains a
negatively charged non-bridging oxygen that could theoretically interact with Ca2+, thus leading
to an increase in catalysis. pNAA was found to only have a 2.64 fold catalysis, which compared
to pNPA's 1.77, is not that significant either. This leads to the belief that Ca2+ catalyzes the
hydrolysis of both pNPA and pNAA by coordinating with the nucleophile, hydroxide, rather than
interacting with the carbonyl oxygen.
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