1
1508 J. Am. Chem. Soc., Vol. 122, No. 46, 2000
Communications to the Editor
Scheme 2. Relative Enzyme Binding Affinities for an
Aldimine in the Ground State and Transition State for
Decarboxylation.
Scheme 1. Decarboxylation of an Amino Acid (R ) H for
glycine) and of Orotidine 5′-phosphate (R )
5-phosphoribosyl-).
plot (Figure 1) that could be extrapolated to yield a rate constant
that it acts as a pure protein catalyst, without the intervention of
metals or other cofactors.
-
17 -1
of ∼2 × 10
( 2) kcal/mol. The half-time for this reaction at 25 °C in neutral
solution is 1.1 billion years, somewhat longer than the half-time
78 million years) reported earlier for the spontaneous decar-
boxylation of orotic acid derivatives in neutral solution (Figure
). Orotidine 5′-phosphate can be considered a modified amino
s
at 25 °C, with an enthalpy of activation of 39
(
Because amino acid decarboxylases depend on cofactors
9
(pyridoxal or pyruvate ) that react covalently with the substrate
(
to form an aldimine intermediate, there is a fundamental difference
in mechanism between the enzymatic and the uncatalyzed
reaction, and the term “transition state affinity” loses it conven-
tional meaning.10 It remains possible, however, to compare the
catalytic effectiveness of such an enzyme with that of the cofactor
alone. Zabinski and Toney (personal communication) have
recently demonstrated that in the absence of enzyme, PLP
catalyzes the decarboxylation of R-aminoisobutyric acid, with a
2
acid (Scheme 1), with its 1-nitrogen atom joined in glycosidic
linkage to ribose 5-phosphate. Despite the slow progress of these
reactions, they proceed much more rapidly than the decarboxy-
lation of glycolic or acetic acids, consistent with the electron-
withdrawing influence of an R-nitrogen atom. In neutral phosphate
buffer, glycolate was decarboxylated to methanol only at tem-
peratures above 300 °C, and acetate had undergone no detectable
decarboxylation after 2 weeks at 360 °C.
-
6 -1
turnover number of 3.9 × 10 s for the aldimine at pH 5.
Comparison of this value with the present rate constant indicates
that PLP alone enhances the rate of decarboxylation by a factor
11
If the value of kcat for the pyridoxal-dependent arginine
of 2 × 10 . As noted in Scheme 2, the much greater rate
-
1 8
decarboxylase of Escherichia coli (1375 s ) is compared with
the present rate constants for spontaneous decarboxylation of
glycine and related amino acids under the same conditions (2 ×
enhancement produced by arginine decarboxylase (k /k ) 7
cat non
19
× 10 ) provides a measure of this protein’s contribution to
8
catalysis. The difference between these factors (3.5 × 10 -fold)
-
17 -1
10
s ), arginine decarboxylase is seen to enhance the rate of
can be considered to set a lower limit on the factor by which the
protein binds the aldimine more tightly in the transition state than
in the ground state for decarboxylation.
19
reaction by a factor (kcat/knon) of 7 × 10 . This rate enhancement
17
somewhat exceeds the rate enhancement (kcat/knon ) 1.4 × 10 )
reported earlier for orotidine 5′-phosphate decarboxylase (ODCase).6
Although these reactions are of comparable difficulty, it should
be noted that ODCase differs from amino acid decarboxylases in
Acknowledgment. We thank R. Zabinski and M. Toney for sharing
results with us before publication and Greg Young for assistance with
NMR. This work was supported by NIH Grant GM-18325, and NIH
Training Grant GM-08570.
(
7) (a) Glycine decarboxylation (this work). (b) R-O-glucoside hydrolysis
(
Wolfenden, R.; Lu, S.; Young, G. J. Am. Chem. Soc. 1998, 120, 6814-
JA002851C
6
815). (c) Fumarate hydration (Bearne, S. L.; Wolfenden, R. J. Am. Chem.
Soc. 1995, 117, 6588-6589). (d) Phosphomonoester and phosphodiester
(9) The decarboxylation of amino acids is the slowest biological process
whose rate appears to have been reported in the absence of a catalyst. When
one considers how such a process might have originated, pyruvic acid seems
more likely than pyridoxal phosphate to have arisen spontaneously as a cofactor
for amino acid decarboxylation under conditions prevailing on the primitive
earth. It may be worth noting that small quantities of pyruvate esters have
been shown to be generated from carbon monoxide and formic acid at high
temperature and pressure in a reducing environment (Cody, G. D.; Boctor,
N. Z.; Filley, T. R.; Hazen, R. M.; Scott, J. H.; Sharma, A.; Yoder, H. S., Jr.
Science 2000, 289, 1337-1340).
hydrolysis (Wolfenden, R.; Ridgeway, C.; Young, G. J. Am. Chem. Soc. 1998,
1
20, 833-834). (e) Mandelate racemization (Bearne, S. L.; Wolfenden, R.
Biochemistry 1997, 36, 1646-1656). (f) Amino acid racemization (Bada, J.
L. J. Am. Chem. Soc. 1992, 94, 1371-1373). (g) Peptide hydrolysis (ref 1b).
(
h) Cytidine deamination (Snider, M. J.; Gaunitz, S.; Ridgway, C.; Short, S.
A.; Wolfenden, R. Biochemistry 2000, 39, 9746-9753). (i) Ribose phos-
phodiester hydrolysis (Thompson, J. E.; Kutateladze, T. G.; Schuster, M. C.;
Venegas, F. D.; Messmore, J. M.; Raines, R. T. Bioorg. Chem. 1995, 23,
4
71-481); and other reactions (ref 6).
8) Blethen, S. L.; Boeker, E. A.; Snell, E. E. J. Biol. Chem. 1968, 243,
671-1677.
(
(10) (a) Lienhard, G. E. Science 1973, 180, 149-154. (b) Wolfenden, R.
Annu. ReV. Biophys. Bioeng. 1976, 5, 271-306.
1