The "neutral" hydrolysis of simple carboxylic esters in water and the rate enhancements produced by acetylcholinesterase and other carboxylic acid esterases

Article abstract of DOI:10.1021/ja204116a

Experiments at elevated temperatures permit the determination of rate constant and thermodynamic activation parameters for the neutral hydrolysis of the neurotransmitter acetylcholine in water. At 25 °C, the extrapolated rate constant for the uncatalyzed (or neutral) hydrolysis of acetylcholine is 3.9 × 10^-7 s^-1 at 25 °C (ΔH^? = 20.0 kcal/mol; TΔS^? = -6.1 kcal/mol). Acetylcholine is more susceptible to neutral and base-catalyzed hydrolysis than ethyl acetate but less susceptible to acid-catalyzed hydrolysis. For acetylcholinesterase from the electric eel, the catalytic proficiency [(k_cat/K _m)/k_neutral] is 2 × 10¹⁶ M^-1, comparable in magnitude with the catalytic proficiencies of aminohydrolases that act on peptides and nucleosides.

Full text of DOI:10.1021/ja204116a

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pubs.acs.org/JACS
The “Neutral” Hydrolysis of Simple Carboxylic Esters in Water and the
Rate Enhancements Produced by Acetylcholinesterase and Other
Carboxylic Acid Esterases
Richard Wolfenden and Yang Yuan*
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599-7260, United States
S Supporting Information
b
their neutral or uncatalyzed counterparts.6 We reasoned that if
ABSTRACT: Experiments at elevated temperatures permit
the determination of rate constant and thermodynamic
activation parameters for the neutral hydrolysis of the
neurotransmitter acetylcholine in water. At 25 °C, the
extrapolated rate constant for the uncatalyzed (or neutral)
hydrolysis of acetylcholine is 3.9 ꢀ 10ꢁ7 sꢁ1 at 25 °C (ΔH‡ =
20.0 kcal/mol; TΔS‡ = ꢁ6.1 kcal/mol). Acetylcholine is
more susceptible to neutral and base-catalyzed hydrolysis
than ethyl acetate but less susceptible to acid-catalyzed
hydrolysis. For acetylcholinesterase from the electric eel,
that were also true of ester hydrolysis, then the rate constant of
the neutral reaction might increase with increasing temperature
relative to the rate constants of the specific acid- and base-catalyzed
reactions (Figure 1), allowing the neutral reaction to emerge as
the predominant mechanism of hydrolysis at elevated tempera-
tures. If rate constants for hydrolysis could be determined with
reasonable precision at elevated temperatures, then it might
be possible to use the Arrhenius relationship to arrive at the
rate constant of the neutral reaction at ordinary temperatures
by extrapolation.
the catalytic proficiency [(kcat/Km)/kneutral] is 2 ꢀ 1016 Mꢁ1
,
We decided to test that possibility by examining the none-
nzymatic hydrolysis of the excitatory neurotransmitter acetylcho-
line (AcCh+) (Scheme 1).
comparable in magnitude with the catalytic proficiencies of
aminohydrolases that act on peptides and nucleosides.
To establish the value of kneut and its dependence on tem-
perature, it was necessary at the outset to have accurate informa-
tion about the rate constants of the acid- and base-catalyzed reac-
tions and their dependence on temperature. To determine the
rate constant (kH+) of the acid-catalyzed reaction in water, we
used 1H NMR to monitor the disappearance of AcCh+ (0.02 M)
in HCl (0.1 N) in experiments conducted over the temperature
range from 10 to 60 °C. For the acid-catalyzed reaction, extra-
polation of an Arrhenius plot of these results yielded kH+ = 3.1 ꢀ
liphatic carboxylic acid esters play central roles in lipid meta-
A
bolism, in the action of excitable tissue, and in the activation
of amino acids for protein biosynthesis. These oxyesters are
rapidly cleaved by hydrolytic enzymes that catalyze either direct
water attack on the ester linkage (such as phospholipase A2)1 or a
double-displacement reaction in which the enzyme and the
substrate form an acyl-enzyme intermediate that subsequently
undergoes hydrolysis (such as acetylcholinesterase).2 At chemical
nerve synapses and neuromuscular junctions, excitation is caused
by release of the neurotransmitter acetylcholine (AcCh+), and its
enzymatic hydrolysis by acetylcholinesterase is required for recov-
ery after excitation. To appreciate the power of these enzymes as
catalysts, it would be useful to have information about the rates of
carboxylic ester hydrolysis in water in the absence of a catalyst.
The kinetics of the specific acid- and base-catalyzed hydrolysis
of oxyesters are relatively well-understood, but the rate constants
of their neutral or “water” reactions (kneutral in eq 1) have remained
somewhat obscure. Discussing earlier efforts3,4 to describe the
neutral reaction of ethyl acetate at 25 °C, Kirsch and Jencks
pointed out that “the experimental difficulties involved in obtaining
(a rate constant) which corresponds to a half-time of 89 years and
represents only 36% of the observed hydrolysis rate under the most
favorable conditions.”5 Thus, the terms in eq 1 that arise from
specific acid and base catalysis [kH+(H+) and kOHꢁ(OHꢁ)] are of
sufficient magnitude to interfere with the direct determination of
kneutral by kinetic measurements under ordinary conditions.
10ꢁ5 Mꢁ1 sꢁ1 at 25 °C (ΔH‡ = 17.4 kcal/mol; TΔS‡
=
ꢁ6.1 kcal/mol). To determine the rate constant (kOHꢁ) of the
base-catalyzed reaction, we used tris(hydroxymethyl)amino-
methane (THAM) buffers (0.1 M, pH 8.1 at 25 °C) over the range
18ꢁ60 °C. THAM was chosen because it is a relatively poor
nucleophile7 and because its enthalpy of ionization (11.35 kcal/mol)
is sufficiently similar to that of water (13.34 kcal/mol)8 to min-
imize the correction required for the effect of changing tempera-
ture on the activity of a hydroxide ion in THAM buffers at constant
pH. At each temperature, rates were extrapolated to zero buffer
concentration to obtain the rate of the base-catalyzed reaction.
An Arrhenius plot of the results was linear and yielded kOHꢁ
=
1.73 Mꢁ1 sꢁ1 at 25 °C(ΔH‡ = 8.6 kcal/mol; TΔS‡ =ꢁ8.5 kcal/mol).
These values are consistent with individual rate constants reported
earlier for AcCh+ hydrolysis at various temperatures.9ꢁ11
At 25 °C, the sum of the values for kH+(H+) and kOHꢁ(OHꢁ)
in eq 1 reaches a minimum value (1.46 ꢀ 10ꢁ9 sꢁ1) at pH 4.6
at 25 °C.
To determine kneutral for AcCh+ at 25 °C, rates of hydrolysis
were determined over the temperature range 78ꢁ118 °C in
potassium acetate buffer (pH 4.6). The heat of ionization of
kobs ¼ kneutral þ kHþðHþÞ þ kOHꢁðOHꢁÞ
ð1Þ
With a few notable exceptions, catalyzed reactions tend to
exhibit lower enthalpies of activation in aqueous solution than do
Received: May 4, 2011
Published: July 27, 2011
r
2011 American Chemical Society
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Table 1. Rate Constants at 25 °C (sꢁ1) and Thermodynamics
of Activation (kcal/mol) for the Hydrolysis of Acetic Acid Esters
T range
Reactant
k25 °C
ΔG‡ ΔH‡ TΔS‡ (°C)
AcCh+, neutral 7.2 ((0.5) ꢀ 10ꢁ9 sꢁ1
28.5 21.0 ꢁ7.5 78ꢁ118
“ ” (H+)
3.1 ((0.2) ꢀ 10ꢁ5 Mꢁ1 sꢁ1 23.5 17.4 ꢁ6.1 10ꢁ60
“ ” (OHꢁ)
1.73 ((0.1) Mꢁ1 sꢁ1
17.1 8.6 ꢁ8.5 18ꢁ60
30.2
EtAc, neutral a (2.5ꢁ5.0) ꢀ 10ꢁ10 sꢁ1
ꢁ
ꢁ
“ ” (H+)b
1.2 ꢀ 10ꢁ4 Mꢁ1 sꢁ1
1.1 ꢀ 10ꢁ1 Mꢁ1 sꢁ1
22.7 15.5 ꢁ7.2
18.7 11.5 ꢁ7.2
“ ” (OHꢁ)b
a The lesser value (2.5 ꢀ 10ꢁ10 Mꢁ1 sꢁ1) is taken from ref 1. The greater
value (5 ꢀ 10ꢁ10 Mꢁ1 sꢁ1) is taken from ref 3 and was estimated from
the intersection of Brønsted plots. b Taken from ref 3 (thermodynamics
of activation not determined).
Figure 1. Influence of pH on an acid- and base-catalyzed reaction in
water, when a neutral reaction is also present. The rate of the neutral
reaction might be expected to increase, relative to the rates of the acid-
and base-catalyzed reactions, with increasing temperature.
Table 2. Rate Enhancements Produced by Oxyesterases at 25 °C
Substrate (enzyme)
knon, sꢁ1
kcat, sꢁ1
kcat/knon
Scheme 1. Hydrolysis of Acetylcholine
AcCh+ (acetylcholinesterase,
EC 3.1.1.7)2
7.2 ꢀ 10ꢁ9
11 700
7.6 ꢀ 1013
diC7PC a (phospholipase A2,
4 ꢀ 10ꢁ10
43
1500
4300
1.1 ꢀ 1011
3.8 ꢀ 1012
1.1 ꢀ 1013
EC 3.1.1.4)
Hydroxybutyrate (hydroxybutyrate 4 ꢀ 10ꢁ10
dimer hydrolase, EC 3.1.1.22)16
Tributyrin (triacylglycerol lipase,
EC 3.1.1.3)17
4 ꢀ 10ꢁ10
a Micellar 1,2-dimyrisotylphosphatidylmethanol (ref 1). In the case of
this micellar substrate, the value of kcat may be limited by physical events
such as lipid exchange between micelles.
each represent 15% of the rate observed. Because the heat of
activation of the neutral hydrolysis of AcCh+ (21.0 kcal/mol) is
larger than the heats of activation of the specific acid- and base-
catalyzed reactions (16.4 and 8.6 kcal/mol respectively), the
modest predominance of the neutral reaction over the acid- and
base-catalyzed reactions at 25 °C becomes more pronounced
with increasing temperature, increasing to 97% of the observed
reaction rate at 100 °C and pH 4.6. That tendency accords with
the hypothesis on which these experiments were based.
The influence of the positively charged guanidinium group of
AcCh+ on the rates of the neutral, acid-catalyzed, and base-
catalyzed reactions can be evaluated by comparison with the
behavior of ethyl acetate. Although the thermodynamics of
activation for the neutral hydrolysis of ethyl acetate have not
yet been established, its approximate rate constant has been
estimated as 2.5 ꢀ 10ꢁ10 sꢁ1 (refs 1 and 2) or 5 ꢀ 10ꢁ10 (ref 3)
at 25 °C. Thus, the neutral hydrolysis of AcCh+ (kneutral = 7.2 ꢀ
10ꢁ9 sꢁ1 at 25 °C) proceeds 15ꢁ30-fold more rapidly than the
neutral hydrolysis of ethyl acetate (k = 2.5 ꢀ 10ꢁ9 sꢁ1 at 25 °C).
The acid- and base-catalyzed reactions of ethyl acetate have been
investigated in greater detail.3ꢁ5 Table 1 shows that AcCh+ is
4-fold less susceptible to acid-catalyzed hydrolysis than ethyl
acetate and 16-fold more susceptible to base-catalyzed hydrolysis.
These differences seem understandable, at least qualitatively, in
terms of the expected retarding influence of a positive charge
on the approach of a nucleophile in the case of the neutral and
base-catalyzed reactions, and on the equilibrium of substrate
protonation in the case of the acid-catalyzed reaction.
Figure 2. Influence of temperature on the rate of neutral hydrolysis of
AcCh+.
acetic acid is small enough (ꢁ0.10 kcal/mol) to render the pH
practically invariant over the temperature range of these experi-
ments. In separate experiments at 80 °C, using potassium acetate
buffer (0.1 M, pH 4.6) with various concentrations of added KCl,
the rate of hydrolysis was found to be insensitive to ionic
strength, decreasing <15% when the ionic strength was raised
from 0 to 3.0 M at 80 °C. At each temperature, the rate of
hydrolysis of AcCh+ was determined at buffer concentrations
ranging from 0.1 to 0.8 M, and the results were extrapolated
to zero buffer concentration. After subtraction of the values of
k
H+(H+) and kOHꢁ(OHꢁ) at that temperature, the resulting
values were plotted as a logarithmic function of absolute tem-
perature, yielding a linear Arrhenius plot (Figure 2). The extra-
polated value of kneutral was 7.2 ꢀ 10ꢁ9 sꢁ1 at 25 °C (ΔH‡ =
21.0 kcal/mol; TΔS‡ = ꢁ7.5 kcal/mol at 25 °C).
These findings are summarized on the first 3 lines of Table 1. At
pH 4.6, where the sum of the rates of the acid- and base-catalyzed
reactions reaches a minimum value at 25 °C,12 the neutral
hydrolysis of AcCh+ represents 70% of the observed rate of
reaction, while the specific acid- and base-catalyzed reactions
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COMMUNICATION
It is of interest to compare the rate constants of these reactions
with those of the corresponding enzymatic reactions. Table 2
(17) Jemel, I.; Fendri, A.; Gargouri, Y.; Bezzine, S. Colloids Surf., B
2009, 70, 238–242.
shows that electric eel acetylcholinesterase (EC 3.1.1.7; kcat
=
11 700 sꢁ1; Km = 9 ꢀ 10ꢁ5 M at pH 7, 25 °C)2 accelerates the
hydrolysis of AcCh+ by a factor of 7.6 ꢀ 1013. Under these
conditions, the value of its catalytic proficiency [(kcat/Km)/kneutral
]
is 1.8 ꢀ 1016 Mꢁ1, comparable in magnitude with the catalytic
proficiencies of aminohydrolases that act on peptides and nucleo-
sides.13,14 Also included in Table 2 are two additional oxyesterases:
phospholipase A2 (EC 3.1.1.4) and a hydroxybutyrate-dimer
hydrolase (EC 3.1.1.22). If ethyl acetate is used as a basis for
comparison, then phospholipase A2 enhances the rate of ester
hydrolysis of a micellar substrate15 by a factor of ∼1011, and
3-hydroxybutyrate-dimer hydrolase16 achieves a rate enhancement
of 4 ꢀ 1012. In view of the magnitudes of the rate enhancements
that they produce, it is evident that both the double displacement
mechanism used by acetylcholinesterase and the mechanism em-
ployed by phospholipase A2 (involving direct water attack) furnish
effective strategies for accelerating carboxylic ester hydrolysis.
’ ASSOCIATED CONTENT
S
Supporting Information. Arrhenius plots for the acidic,
b
basic, and neutral hydrolysis of acetylcholine. This material is
available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
water@med.unc.edu
’ ACKNOWLEDGMENT
We thank the National Institutes of Health (Grant No.
GM-18325) for supporting this work.
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