Phospholipase C Mechanism
A R T I C L E S
bridging interactions through nonbridging O-S replacement
should bring about a similar consequence.
it has been known to catalyze cleavage of phosphatidylinositol
analogues with double or single chain leaving groups and
hydrophobic or hydrophilic leaving groups.22 Additional inspira-
tion to conduct the LFER study on PI-PLC was our earlier
finding of strong resistance of alkyl inositol phosphates to
chemical cyclization as compared to analogous alkyl ribonucle-
otides, suggesting different mechanism, as well as the relative
paucity of LFER data pertinent to phosphodiester cleavage (other
than RNase reactions). Furthermore, application of the Brønsted-
type relationship should provide further evidence supporting
cooperativity of catalytic functions of Arg69, Asp33, and His82
in their interaction with the nonbridging and leaving group
oxygen atoms.
The extent of the negative charge development on the leaving
group oxygen in the transition state for phosphate ester
hydrolysis can be estimated by employing a Brønsted-type linear
free energy relationship (eq 1), by examining rate constants of
the cleavage reaction as a function of varying pKa values of the
leaving group.11-17
log k ) âlg pKa + C
(1)
where k is the rate constant for P-O bond cleavage.
The strong relationship (high negative âlg), indicating a large
amount of the negative charge localized on the leaving group,
suggests a transition state with negatively charged leaving group
(hence resembling the ionized alcohol product), whereas the
flat response (low negative âlg) indicates mechanism with such
charge absent. It is important to stress, however, that unlike in
the nonenzymatic reactions, where the Brønsted coefficient is
directly related to the degree of bond formation or bond
breaking, enzymatic reactions pose a more complex case. In
enzyme reactions, protonation of a leaving group by a general
acid function can reduce its negative charge, resulting in a weak
response of reaction rates to pKa change, despite possibly a
significant degree of ester bond breaking.14,15 Before any
mechanistic interpretation of the dependence of the rate constants
vs leaving group pKa is attempted, it is also essential to ascertain
that the cleavage of the P-O bond is a rate-limiting step. Only
in such case, the differences in the stability of transition states
(TS) brought upon by the variation in the stability of the leaving
group (pKa) will be manifested in different rate constants for
the cyclization reactions. For example, observation of a weak
response of rate constants upon pKa variation can be taken as
evidence that very little negative charge is developed on the
leaving group in TS. On the other hand, such flat relationship
would also be expected if the physical step was rate-determining,
since minor changes in the leaving group structure would not
have a significant effect on the rates of physical processes such
as conformational changes of enzyme, kinetics of binding steps,
etc. Once the chemical step rate limitation is established, the
comparison of LFER coefficients for a reaction series of
different types of substrates should still be a valuable tool in
exploring catalytic significance of interactions of the phosphate
group with enzyme residues.
Materials and Methods
Synthesis of Alkyl and Aryl Inositol Phosphates: General
Procedure (Scheme 1, Supporting Information). 2,3,4,5,6-Pentakis-
(methoxymethylene)-myo-inositol23 (1, 1 mmol) was dissolved in
anhydrous diisopropylethylamine (2.5 mmol) and a minimum volume
of anhydrous chloroform. The solution was cooled to -78 °C, and
O-methyl phosphorodichloridite (1.25 mmol) was added dropwise via
a syringe under nitrogen. The reaction mixture was vigorously stirred
and warmed to room temperature over a period of 6 h. The solution of
the second alcohol (2 mmol) in dry chloroform (5 mL) was added
slowly via a cannula at 0 °C. The reaction mixture was warmed to
room temperature and stirred for 12 h. The phosphite triester 2 was
oxidized by addding tert-butyl hydroperoxide (2 mmol, 5 M solution
in decane) via a syringe or sulfurized with elemental sulfur (2 equiv)
in anhydrous carbon disulfide (12 h at room temperature). The fully
protected phosphate 3a or phosphorothioate 3b was treated with
anhydrous neat trimethylamine (1 mL) at 50°C for at least 24 h. The
progress of the demethylation reaction was monitored by 31P NMR.
After the reaction had been completed, trimethylamine was removed
under vacuum and the crude product was subjected to final deprotection
as follows: Into the phosphate or phosphorothioate diester product from
the above trimethylamine reaction was added ethanethiol (1 mL) and
boron trifluoride-etherate (100 µL). The reaction mixture was stirred
at room temperature for 1 h, and the reaction was monitored by TLC.
Ethanethiol was removed under vacuum, and the residue was chro-
matographed on silica gel using elution with chloroform-methanol-
NH4OH to yield pure deprotected phosphodiesters 4. This method was
applied to all long-chain O-alkyl inositol phosphates. Short-chain alkyl
phosphodiesters were dissolved in 50% aqueous acetic acid, the obtained
solution was heated at 50-60 °C, and the reaction progress was
monitored by 31P NMR. The reaction time varied from 30 h to a few
days, and the reaction provided a pure product 4 in most cases.
Additional purifications were performed by anion-exchange chroma-
tography on Dowex 1×8 200 anion-exchange resin using elution with
ammonium formate step gradient. The aryl esters 3a,b were obtained
analogously as the alkyl esters, except the phosphites 3 were oxidized
with tetrabutylammonium periodate at -20 °C. The aryl esters 3a,b
were purified by chromatography on silica gel using hexane-acetone
(6:1, v/v) to afford pure products with 60-70% yield as colorless oils.
The deprotection of aryl esters 3a,b was performed analogously as
described for long-chain aliphatic esters, and the crude products 4 were
purified by chromatography on silica gel using chloroform-methanol-
acetic acid (6:4:0.1, v/v) as the eluent to yield pure aryl esters 4 as
colorless or pale yellow solids. Physical characteristics of each
synthesized compound are listed in the Supporting Information.
One of the requirements for conducting LFER for an enzyme
reaction is that the catalytic site accommodate a variety of
structures modified such as to alter the pKa values.16 This
condition is difficult to realize with enzymes displaying strong
substrate specificity. Phosphatidylinositol-specific phospholipase
C is well suited for this approach, since although it is absolutely
specific with respect to the structure of inositol phosphate,18-21
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