COMMUNICATION
pubs.acs.org/JACS
Kinetic Challenges Facing Oxalate, Malonate, Acetoacetate,
and Oxaloacetate Decarboxylases
Richard Wolfenden,* Charles A. Lewis, Jr., and Yang Yuan
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599-7260, United States
S Supporting Information
b
Table 1. Rate Constants at 25 °C (knon, sꢁ1) and Thermo-
ABSTRACT: To compare the powers of the corresponding
enzymes as catalysts, the rates of uncatalyzed decarboxyla-
tion of several aliphatic acids (oxalate, malonate, acetoace-
tate, and oxaloacetate) were determined at elevated temper-
atures and extrapolated to 25 °C. In the extreme case of
oxalate, the rate of the uncatalyzed reaction at pH 4.2 was 1.1
ꢀ 10ꢁ12 sꢁ1, implying a 2.5 ꢀ 1013-fold rate enhancement
by oxalate decarboxylase. Whereas the enzymatic decarbox-
ylation of oxalate requires O2 and MnII, the uncatalyzed
reaction is unaffected by the presence of these cofactors and
appears to proceed by heterolytic elimination of CO2.
dynamic Parameters of Activation (kcal/mol) for the Un-
catalyzed Decarboxylation of Oxaloacetate, Acetoacetate, and
Malonate at pH 6.8 and Oxalate at pH 4.2; Values for the Pure
Monoanions Are Given in Italics (Also See the Supporting
Information)
reactant (product)
knon
ΔGq ΔHq TΔSq T range (°C)
oxaloacetate (pyruvate) 2.8 ꢀ 10ꢁ5 23.6 17.2 ꢁ6.4
9.2 ꢀ 10ꢁ5 24.2 17.2 ꢁ7.0
23ꢁ70
acetoacetate (acetone) 3.0 ꢀ 10ꢁ7 26.2 23.5 ꢁ2.7
3.0 ꢀ 10ꢁ7 26.2 23.5 ꢁ2.7
23ꢁ61
malonate (acetate)
1.2 ꢀ 10ꢁ10 30.9 30.0 ꢁ0.9
1.5 ꢀ 10ꢁ9 29.4 30.0 þ0.6
1.1 ꢀ 10ꢁ12 33.7 26.9 ꢁ6.8
1.8 ꢀ 10ꢁ12 33.3 26.9 ꢁ6.6
80ꢁ130
150ꢁ190
oxalate (formate)
o be useful at the limited concentrations at which they are
T
present within cells (<10ꢁ4 M),1 enzymes must act rapidly
on their substrates. But in the absence of enzymes, biological
reactions proceed with half-lives ranging from <1 min for the
dehydration of bicarbonate2 to >1 billion years for the decarbox-
ylation of glycine.3 Those rate enhancements are of interest in
estimating the power of enzymes and artificial catalysts and their
expected sensitivities to transition state analogue inhibitors. Here
we compare the rates of spontaneous and enzymatic decarbox-
ylation of oxalate with those of malonate, acetoacetate, and
oxaloacetate.
of malonate and acetoacetate were estimated by monitoring the
disappearance of the reactants, and each reaction followed simple
first-order kinetics to completion. In the cases of oxaloacetate
(whose CꢁH protons exchange rapidly with solvent water)
and oxalate (with no carbon-bound protons), the rates were
estimated by monitoring the appearance of the corresponding
decarboxylation products, pyruvate and formate. At each tem-
perature, the heating times (between 2 and 72 h) were chosen to
allow consumption of the reactant to reach between 15 and 85%
completion, yielding individual rate constants with estimated
errors of (3%. These rate constants plotted as a logarithmic
function of 1/T (with T in K) showed a linear relationship over the
full range of temperatures examined, which was used to estimate the
enthalpy of activation (ΔHq) and the rate constant for each
uncatalyzed reaction at 25 °C (knon). The results are shown in
Table 1, and they are included in further detail in the Supporting
Information along with values previously reported for these and
other decarboxylation reactions.
The observed rate constants for the decarboxylation of the mono-
anions of oxaloacetic, acetoacetic acid, and malonic acid mono-
anions fall close to a linear Brønsted plot based on the pKa values of
the carbon acids produced by decarboxylation (Figure 1), yielding a
slope (β = ꢁ0.7) consistent with the development of substantial
negative charge at the site where CO2 elimination occurs.
Enzymes use various strategies to catalyze these decarboxyla-
tion reactions, employing an imine-forming lysine residue in the
case of acetoacetate or a divalent cation (MgII, MnII, ZnII, or CoII)
Kinetic experiments on the monoanions of malonate, acet-
oacetate, and oxaloacetate were conducted in potassium phos-
phate buffer (pH 6.8), where the corresponding decarboxylases
are maximally active.4ꢁ6 The nonenzymatic decarboxylation of
oxalate was examined in potassium acetate buffer (pH 4.2),
because oxalate decarboxylase is maximally active near pH 4.2.7
Phosphate and acetate buffers were chosen because acetic acid
and the phosphoric acid monoanion (like the acids undergoing
decarboxylation) exhibit near-zero (<1 kcal/mol) heats of pro-
ton dissociation,8 canceling the effects of varying temperature on
the state of ionization of each substrate. Samples of the potassium
salt of each acid (0.01 M) in potassium acetate or phosphate
buffer (0.1 M) were introduced into quartz tubes, sealed under
vacuum, and placed in convection ovens for various intervals at
temperatures maintained within (1.5 °C as indicated by ASTM
thermometers. For each acid, the range of temperatures exam-
ined is indicated in Table 1. After the samples were cooled, they
were diluted with D2O containing pyrazine (5 ꢀ 10ꢁ4 M), which
1
was added as an integration standard. In each case, H NMR
analysis showed quantitative conversion of the carboxylic acid to
the expected product of decarboxylation. The rates of decarboxylation
Received: December 24, 2010
Published: March 24, 2011
r
2011 American Chemical Society
5683
dx.doi.org/10.1021/ja111457h J. Am. Chem. Soc. 2011, 133, 5683–5685
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