Catalysis by desolvation: The catalytic prowess of SAM-dependent halide-alkylating enzymes

Article abstract of DOI:10.1021/ja406381b

In the biological fixation of halide ions, several enzymes have been found to catalyze alkyl transfer from S-adenosylmethionine to halide ions. It proves possible to measure the rates of reaction of the trimethylsulfonium ion with I^-, Br^-<

Full text of DOI:10.1021/ja406381b

Communication
pubs.acs.org/JACS
Catalysis by Desolvation: The Catalytic Prowess of SAM-Dependent
Halide-Alkylating Enzymes
†
§
Danielle C. Lohman, David R. Edwards, and Richard Wolfenden*
Department of Biochemistry and Biophysics, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599,
United States
*
S Supporting Information
pure protein catalysts that act on their substrates without the
assistance of metals or other cofactors.
4
ABSTRACT: In the biological fixation of halide ions,
several enzymes have been found to catalyze alkyl transfer
from S-adenosylmethionine to halide ions. It proves
possible to measure the rates of reaction of the
In alkyl transfer from a sulfonium ion to a halide ion, general
acid−base catalysis is improbable or impossible. To appreciate
the kinetic barriers that are surmounted by enzymes of this
kind, it would be desirable to compare the rates of these
enzyme-catalyzed reactions with the rates of the uncatalyzed
reactions in water in the absence of a catalyst. That information
would also be expected to be useful in calibrating alternative
approaches, such as QM/MM, to the simulation of enzyme rate
enhancements based on structural information. In the experi-
−
−
−
−
−
trimethylsulfonium ion with I , Br , Cl , F , HO , and
H O in water at elevated temperatures. Comparison of the
2
resulting second-order rate constants, extrapolated to 25
°
C, with the values of k /K reported for fluorinase and
cat m
chlorinase indicates that these enzymes enhance the rates
1
5
of alkyl halide formation by factors of 2 × 10 - and 1 ×
1
17
0 -fold, respectively. These rate enhancements, achieved
ments described here, we sought to obtain that information
without the assistance of cofactors, metal ions, or general
acid−base catalysis, are the largest that have been reported
for an enzyme that acts on two substrates.
+
using the symmetrical trimethylsulfonium ion (Me S ) as an
3
5
alkyl donor.
In these kinetic experiments, reaction mixtures containing
+
−
−
−
−
Me S :BF (0.02 M) and the potassium salt of I , Br , Cl ,
3
4
−
−
F , or HO (0 to 1.0 M) were sealed under vacuum in quartz
tubes and incubated at temperatures ranging from 65 to 200 °C
in ovens equipped with ASTM thermometers. After various
time periods, the reaction was stopped by cooling, and the
n the biological fixation of halide ions, several enzymes have
been found to catalyze alkyl transfer from S-adenosylme-
I
thionine to halide ions. Among these enzymes are a fluorinase
1
(
FDAS, EC 2.5.1.63) from Streptomyces cattleya and a
product mixture was diluted 5-fold with D O containing added
2
chlorinase (SalL, EC 2.5.1.94) from the marine bacterium
2
pyrazine as an integration standard. Analysis of product
Salinispora tropica, which catalyze attack by halide ions on S-
1
+
+
mixtures by H NMR showed conversion of Me S to methanol
adenosylmethionine (SAM ) to generate 5′-halogenated
3
and dimethyl sulfide, with no other detectable products (Figure
derivatives of adenosine (Scheme 1A). Another group of
halide-fixing enzymes, that are probably responsible for the
appearance of halomethanes in the atmosphere, catalyzes a
S1). The progress of reaction was followed by monitoring the
+
decline of the integrated signal intensity of Me S , which was
3
+
closely matched in each case by the appearance of methanol.
The halomethane products of these reactions were not
observed and were not expected to accumulate because they
are hydrolyzed at least 100-fold more rapidly than the
decomposition of Me3S+ under the conditions of these
halide attack at the S-methyl group of SAM , with displacement
3
of S-adenosylhomocysteine (Scheme 1B). These enzymes are
Scheme 1. Reactions Catalyzed by Two Groups of Cofactor-
Independent Halogenases
−
experiments. Thus, the rate constant for attack by Cl in the
present experiments was 1.0 × 10−
5
M
−1 −1
s
at 150 °C, where
−3
−1
6
the rate constant for CH Cl hydrolysis is 4.4 × 10 s .
3
In Figure 1, apparent first-order rate constants for the
+
decomposition of Me S at 150 °C are plotted as a function of
3
the concentration of NaCl. The intercept on the vertical axis
corresponds to kwater = (8 ± 2) × 10−
7
s
−1
at 150 °C, in
satisfactory agreement with the rate constant of (8.6 ± 0.9) ×
−7
−1
+
1
0
s
observed for decomposition of Me S in the absence of
3
added chloride ion (Figure S4). Second-order rate constants
were corrected by subtracting the rate of background hydrolysis
(
kwater, always <10% of the total rate) from the observed rate of
Received: July 2, 2013
Published: September 16, 2013
©
2013 American Chemical Society
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Journal of the American Chemical Society
Communication
Table 1. Rate Constants and Thermodynamics of Activation
+
for Reactions of Nucleophiles with Me S in Water at 25 °C,
3
+
Compared with Rates of Enzymatic Methylation with SAM
‡
+
ΔH
k /K
cat
(M
m(SAM )
s
k (M− s−1
1
)
−1 −1
RE
5 × 1014
a
Nu
kcal/mol
)
2
I−
−11
−12
−13
−13
−10
b
3.1 × 10
3.2 × 10
3.7 × 10
1.9 × 10
2.9 × 10
5.8 × 10−
30.3
31.9
34.5
5000
Br−
13 300
b
b
1.2 × 10
1.2 × 10
2.8 × 10
NA
16
Cl−
15 000
17
15
F−
c
d
32.4
179
HO−
32.5
38.7
NA
17
e
20
H O
2
7500
1.8 × 10
a
+
Figure 1. Decomposition of Me S in water at 150 °C, plotted as a
Values of these rate enhancements (RE) were obtained by dividing
+
3
the second-order rate constant k /K for the enzyme reaction,
m(SAM )
in the presence of saturating halide ion, by one-third of the second-
order rate constant (k ) of the uncatalyzed reaction evaluated in the
present work (see Table S1 for binding constants). Chlorinase (ref
function of the concentration of NaCl at constant ionic strength (1.0,
maintained by addition of NaBF4).
cat
2
b
decomposition and dividing that result by the concentration of
halide or hydroxide ion.
2
). Although it has been described as a “chlorinase,” Sa1L is almost
c
equally active as a brominase or an iodinase (ref 2). Considerably
lower values (22.0 or 26.8 kcal/mol) have been estimated on the basis
of QM/MM simulations (ref 7). Fluorinase (ref 8). Obtained by
dividing the observed first-order rate constant (k ) for enzymatic
hydrolysis of SAM by the hydrolase DUF62 (ref 9) by the molarity of
water.
Activation parameters were obtained by fitting those second-
order rate constants, calculated from the results obtained at 6 or
more temperatures over the range from 65 to 200 °C, to the
Arrhenius equation. Rate constants at 25 °C were estimated by
d
e
cat
+
extrapolation of linear plots of k as a logarithmic function of
2
the reciprocal of absolute temperature (Figure 2) (see also
K ) reported for SAM in the presence of saturating nucleophile
m
8
2
indicates that fluorinase and chlorinase enhance the rates of
15
17
alkyl halide formation by factors of 2.4 × 10 - and 1.2 × 10 -
14
fold, respectively, at 25 °C. If we assume that substrate water
is not saturating, as appears to be the case for other hydrolytic
15
+
enzymes, then the rate of hydrolysis of SAM is enhanced by
the DUF62 enzyme to an even greater extent (1.8 × 10 -
fold). For steric reasons, more elaborate sulfonium ions might
9
20
16
+
be expected to react somewhat more slowly than Me S . If that
3
is the case, then the rate enhancements achieved by these
enzymes are even larger than these values. Moreover, they
exceed rate enhancements that have been reported for other
bisubstrate reactions that have been studied in this way, which
include several O-phosphorylating kinases (ranging from 3 ×
1
2
14
17
1
0 - to 5 × 10 -fold) and the peptidyltransferase center of
7
18
+
Figure 2. Decomposition of Me S in water in the presence of NaCl
3
the ribosome (3 × 10 -fold).
What is the source of the large rate enhancements produced
(
1.0 M) plotted as a function of absolute temperature over the range
−13
from 90 to 150 °C. Linear regression yields k = 3.7 (±1.5) × 10
M
kcal/mol at 25 °C.
2
−1
−1
‡
‡
by chlorinase and fluorinase? In the reported crystal structures
s , with ΔH = 34.5 (±1.5) kcal/mol and TΔS = 0.2 (±2.0)
1
2
of fluorinase and chlorinase, water is stripped from the halide
ion as it is bound; the halide ion’s remaining contacts with
+
water vanish when SAM is bound during formation the ternary
Figures S4−S8). The resulting second-order rate constants and
complex, and the halide ion is held in a position appropriate for
+
+
activation parameters for the decomposition of Me S in the
in-line attack on SAM . In general, desolvation is known to
3
presence of halide ions, hydroxide ion, and water are shown in
Table 1.
enhance the rates of reactions in which charge is delocalized or
19
neutralized in the transition state, and simulations indicate
−
In accord with earlier studies of nucleophilic substitution in
that only partial desolvation is required for F to serve as a
1
0,11
+
powerful nucleophile. Halide ions and Me3S+ have been
shown to exist predominantly as ion pairs in organic solvents,
20
water
and with earlier evidence that Me S decomposes by
3
12
an S 2 mechanism at 100 °C in ethanol, a logarithmic plot of
N
2
1,22
k as a function of nucleophilicity indicates that halide reactions
such as acetone and tetrachloroethane.
When we examined
2
+
+ −
with Me S involve strong nucleophilic participation (Figure
the decomposition of Me S :I (0.02M) in DMSO-d we found
3
3
6
S9). Structural and kinetic evidence suggests that enzymatic
that this reaction followed first-order kinetics up to at least 90%
alkyl transfer to halide ions likewise proceeds by S 2
completion, consistent with decomposition of ion-paired
N
1
3
+ −
substitution.
Me S :I to dimethyl sulfide and iodomethane. This reaction
3
4
In free solution, nucleophilic attack is equally likely to occur
was found to proceed 4 × 10 -fold more rapidly in DMSO than
+
at each of the three methyl groups of Me S , whereas only a
in water at 37 °C (Figure S9). In surroundings less polar than
DMSO, the accelerating effects of removing the substrates from
water might approach the much larger solvent effects that have
been observed for the hydrolysis of phosphate esters in
3
single alkyl group is transferred in the enzymatic reactions of
+
SAM . Division of the observed second-order rate constants
(k2) in Table 1, reduced by a factor of 3 to incorporate that
23
statistical correction, into the second-order rate constants (kcat/
cyclohexane.
1
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Journal of the American Chemical Society
Communication
In summary, the relative resistance of the trimethylsulfonium
ion to hydrolysis makes it possible to measure rates of methyl
(9) Deng, H.; McMahon, S. A.; Eustaq
Naismith, J. H.; O’Hagan, D. ChemBioChem 2009, 10, 2455.
(10) Pearson, R. G.; Sobel, H.; Songstad, J. J. Am. Chem. Soc. 1968,
́
uio, A. S.; Moore, B. S.;
−
−
−
−
transfer to I , Br , Cl , and F , in water at elevated
temperatures. The resulting second-order rate constants,
obtained by extrapolation of Arrhenius plots to 25 °C, imply
that fluorinase and chlorinase enhance the rates of alkyl halide
9
0, 319.
(
(
(
11) Swain, C. G. S.; Scott, C. B. J. Am. Chem. Soc. 1953, 75, 246.
12) Pocker, Y.; Parker, A. J. J. Org. Chem. 1966, 31, 1526.
13) Cadicamo, C. D.; Courtieu, J.; Deng, H.; Meddour, A.;
1
5
17
formation by factors of 2 × 10 - and 1 × 10 -fold,
respectively. These rate enhancements, achieved without the
assistance of metals, cofactors, or general acid−base catalysis,
give some indication of how much can be accomplished by
juxtaposition and desolvation of two substrates at an enzyme’s
O’Hagan, D. ChemBioChem 2004, 5, 685.
(
14) Eustaquio, A. S.; Harle, J.; Noel, J. P.; Moore, B. S.
́
ChemBioChem 2008, 9, 2215.
(15) Dzingeleski, G. D.; Wolfenden, R. Biochemistry 1993, 32, 9143.
(16) In the case of catecholamine O-methyltransferase, Schowen and
his associates estimated a rate enhancement of 3 × 10 -fold at 37 °C:
Mihel, I.; Knipe, J. O.; Coward, J. K.; Schowen, R. L. J. Am. Chem. Soc.
2
4
16
active site. It is also possible that, following substrate binding,
the reactants are compressed as they proceed toward the
transition state, as has been demonstrated in the case of
1
979, 101, 4349.
16
(17) Stockbridge, R. B.; Wolfenden, R. J. Biol. Chem. 2009, 284,
catechol O-methyltransferase.
2
2747.
(
(
(
18) Schroeder, G. K.; Wolfenden, R. Biochemistry 2007, 46, 4037.
19) Hughes, E. D. I.; Ingold, C. K. J. Chem. Soc. 1935, 244.
20) Vincent, M. A.; Hillier, I. H. Chem. Commun. 2005, 5902.
ASSOCIATED CONTENT
■
*
S
Supporting Information
H NMR spectrum of a reaction mixture. Plots of kobs vs
1
(21) Swain, C. G. B.; Burrows, W. D.; Schowen, B. J. J. Org. Chem.
1
968, 33, 2534.
[
NaBF ] and [HCl]. Arrhenius plots for the decomposition of
4
(
(
22) Swain, C. G. K.; Kaiser, L. E. J. Am. Chem. Soc. 1958, 80, 4089.
23) Stockbridge, R. B.; Wolfenden, R. Chem. Commun. 2010, 46,
+
−
−
−
−
Me S with H O, F , Br , I , and HO . Plot of log(k ) vs
3
2
2
nucleophilicity constant for the halides. A plot of decom-
4
306.
+
−
position of Me S I in DMSO-d at 50 °C. This material is
3
6
(24) For a lucid review of these issues: Schowen, , R. L. Transition
States of Biological Processes; Fandour, , R. D., Schowen, , R. L., Eds.;
Plenum Press: New York, 1978,; pp 77−−114.
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
water@med.unc.edu
Present Addresses
†
Department of Biochemistry, University of Wisconsin at
Madison, Madison, WI 53706.
§
Afton Chemical Corporation, Richmond, VA 23219.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by National Institutes of Health
Grant #GM-18325.
REFERENCES
■
(
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(
4) Products of methylation by SAM are limited by the nature of its
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II
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2
(
005, 102, 1011 and references cited therein).
+
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+
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3
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dx.doi.org/10.1021/ja406381b | J. Am. Chem. Soc. 2013, 135, 14473−14475