A selective chemical probe for coenzyme A-requiring enzymes

Article abstract of DOI:10.1002/anie.200702485

(Chemical Equation Presented) Tagging transferases: A coenzyme A (CoA)-based affinity probe with a sulfoxycarbamate functionality can selectively identify several acetyltransferases relative to other enzymes and proteins. It leaves behind a desthiobiotin tag that can be used for western blotting and mass spectrometric characterization (see picture; Nu = nucleophile).

Full text of DOI:10.1002/anie.200702485

Angewandte
Chemie
DOI: 10.1002/anie.200702485
Affinity Probes
A Selective Chemical Probe for Coenzyme A-Requiring Enzymes**
Yousang Hwang, Paul R. Thompson, Ling Wang, Lihua Jiang, Neil L. Kelleher, and
Philip A. Cole*
Structural and functional annotation of the human proteome
is one of the critical challenges in biomedical research. Recent
chemical approaches have been brought to bear on system-
atically interrogating enzymes involved in protein phosphor-
ylation, proteolysis, lipid modification, glycosylation, deimi-
nation, methyl transfer, and acetylation.[1] An elegant strategy
for analyzing substrates of acetyltransferases by using chlor-
oacetyl coenzyme A (CoA) has been reported.[1q] However,
to our knowledge, no chemical probes have yet been
established to specifically tag CoA-dependent proteins by
covalent cross-linking. These probes could be especially
useful in the identification and analysis of histone and protein
acetyltransferases, which contain many families with limited
homology.[2] Herein, we describe the synthesis and functional
analysis of a new CoA chemical probe that shows selectivity
in tagging acetyltransferases.
There are no absolutely conserved catalytic residues
throughout the different groups of acetyltransferases,
although there are elements of phosphopantetheine binding
that appear to be structurally conserved across many of these
enzymes.[2] Consequently, we designed two probes to inter-
rogate acetyltransferases which exploit the CoA binding
pocket. The fluoroacetonyl CoA 1 (Scheme 1) was envisaged
to be a mimic of acetyl CoA and take advantage of the known
reactivity of a-halocarbonyl compounds toward nucleophilic
attack. 32P-labeled 1 produced enzymatically[3] (Scheme S1 in
the Supporting Information) was examined as an affinity
agent against a series of acetyltransferases and control
proteins.
As can be seen by autoradiography (Figure S1 in the
Supporting Information), compound 1 could selectively label
two acetyltransferases (p300[4] and arylalkylamine N-acetyl-
transferase (AANAT)[2]) but not a third (p300/CBP-associ-
ated factor (PCAF)[2]). Although 1 did not label two non-
acetyltransferases, 14-3-3[5] or low-molecular-weight phos-
Scheme 1. Strategy for labeling proteins based on CoA affinity.
Nu=nucleophile.
photyrosine phosphatase (LMW-PTP)[6], 1 did label two
protein kinases, Csk[7] and Src.[7] Compound 1 did not perform
well in cellular extracts (Figure S1 in the Supporting Infor-
mation) and in competition experiments with acetonyl CoA
(actCoA; Figure S1 in the Supporting Information).
Given the limitations of 1, we turned to a different
electrophilic probe 2 (Scheme 1), which we hoped could be
more useful. Compound 2 was designed to have the potential
to introduce a desthiobiotin moiety at the protein modifica-
tion site, and the covalent desthiobiotin could then be used as
an affinity handle (Scheme 1). Although to our knowledge a
thiocarbamate sulfoxide functionality has not previously been
used in protein modification, it was envisaged that this group
could be attacked by enzyme nucleophiles, especially cys-
teines, at the carbonyl carbon atom,[8] thus generating
carbamylated targets and liberating CoA sulfenic acid.
Synthesis of compound 2 was carried out as shown in
Scheme 2. The starting Boc diamine 3 was bilaterally
derivatized to install the thiocarbamate and desthiobiotin
groups. Oxidation of the thiocarbamate with excess Oxone
led to sulfone 5, which was used in trans acylation with
CoASH. Controlled oxidation of the thiocarbamyl moiety of
the penultimate intermediate with Oxone resulted in suc-
cessful conversion to the desired target compound 2. As
hoped, 2 proved to be reasonably stable under physiological
conditions and resistant to reaction with low concentrations
of thiols.
[*] Dr. Y. Hwang, Dr. P. R. Thompson, L. Wang, Prof. Dr. P. A. Cole
Department of Pharmacology and Molecular Sciences
Johns Hopkins School of Medicine
Baltimore, MD 21205 (USA)
Fax: (+1)410-614-7717
E-mail: pcole@jhmi.edu
L. Jiang, Prof. Dr. N. L. Kelleher
Department of Chemistry
University of Illinois
Champaign-Urbana, IL 61801 (USA)
[**] We are grateful for financial support from the NIH, the Kaufman
Foundation, and to Cole laboratory members for advice and
discussion.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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Scheme 2. Synthesis of sulfoxide 2. a) Ethyl thiochloroformate
Figure 1. A) In vitro labeling experiments on non-CoA-requiring pro-
teins with 2 (25 mm). B) In vitro labeling experiments on CoA-requiring
proteins with 2 (25 mm) and competition by actCoA (3 mm). C) In vitro
labeling of cell lysates with 2 (50 mm). The lysates prepared from HeLa
nucleus (NE) were either spiked with recombinant p300-histone
acetyltransferase (p300-HAT; approximately 1/50) or not spiked.
D) Labeled cell lysates were probed by western blotting with anti-HAT1
antibody after streptavidin–agarose purification (capture). BSA=bo-
vine serum albumin, ESA=essential SAS2-related acetyltransferase 1,
GCN5=growth-control nonrepressed 5.
(ClCOSC2H5), 08C, aqueous NaOH/diethyl ether; b) 6n HCl, MeOH,
RT; c) TSTU, desthiobiotin, DIPEA, RT, dioxane/H2O/DMF; d) Oxone,
RT, MeOH/H2O; e) CoASH, LiOH, MeOH/H2O; f) Oxone, 08C,
MeOH/H2O then reversed-phase column chromatography. Boc=tert-
butoxycarbonyl, TSTU=N,N,N’,N’-tetramethyl-O-(N-succinimidyl)ura-
nium tetrafluoroborate, DIPEA=ethyldiisopropylamine.
We examined the reactivity of 2 with a number of protein
targets and a range of acetyltransferases, as well as with other
enzymes and proteins (Figure 1). Compound 2 showed
efficient labeling of each of the acetyltransferases tested
(p300, PCAF, AANAT, GCN5,
ESA[2]) and in each of these cases,
labeling was effectively competed by
excess actCoA (Figure 1B). Com-
pound 2 did not show significant
labeling of 14-3-3, PTP, or Csk (Fig-
ure 1A). Src was also labeled by 2,
but this labeling was not significantly
competed by actCoA (Figure 1A,B),
which suggests that 2 is far more
selective in targeting acetyltransfer-
ases than 1.
tallographically and contains an active-site cysteine with the
potential to attack acetyl CoA.[2] The structure of p300 has not
An advantage of 2 is that the
desthiobiotin left behind can be used
for pull-down experiments. This was
hypothesized to be useful in the
context of localizing modifications
in combination with proteolytic
digestion. We attempted such experi-
ments for two different acetyltrans-
ferases, yESA1 and p300 (Figure 2).
Figure 2. A) MALDI MS analysis of the streptavidin-enriched peptides from p300-HAT and yESA1
after labeling with 2 and tryptic digestion. B) Labeled peptides and confirmation of labeling site
from p300-HAT by MS/MS analysis. Upward and downward slashes represent the observed c and zC
ions, respectively. All the c or zC ions in red are modified while all the c or zC ions in blue are not
modified, which leads to the localization of desthiobiotinylation to residues colored in yellow. The
yESA1 has been characterized crys- two lines in (B) correspond to yESA1 (top) and p300-HAT peptides (bottom).
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Angewandte
Chemie
Proteins were separated by SDS-PAGE and transferred to nitro-
been reported, and its sequence is not homologous to those of
other HATs.[4]
cellulose membranes. The membranes were blocked for 1 h with 5%
BSA in Tris-buffered saline (TBS) with 0.1% Tween 20 (TBST) at
room temperature, followed by incubation for 1 h with horseradish
peroxidase streptavidin in TBST. After four washes with changes
every 15 min in TBST, the biotinylated proteins were visualized by
enhanced chemiluminescence.
Trypsin digestion, purification, and detection of biotinylated
peptides: The labeled proteins (1 mm of p300-HAT or yESA1 treated
with 25 mm 2 as described above) were dialyzed against TBS to
remove 2. The dialysate was digested overnight at 378Cwith trypsin,
then incubated with streptavidin–agarose beads for 1 h at room
temperature. The beads were washed three times with ten volumes of
low-salt wash buffer (TBS: 50 mm Tris pH 7.4, 150 mm NaCl), three
times with high-salt wash buffer (50 mm Tris pH 7.4, 500 mm NaCl),
and finally with ten volumes of dd H2O (twice). After washing, the
beads were eluted with 30% aqueous acetonitrile and 0.5% trifluoro-
acetic acid. The eluted peptides were partially dried by vacuum
centrifugation and analyzed by either MALDI-TOF or liquid
chromatography–MS/MS.
After labeling, yESA1 and p300 were treated with trypsin
and the peptide digests purified with streptavidin. Mass
spectrometric (MS) analysis of the purified peptides from
yESA1 (Figure 2A, right) gave two major peaks (m/z 2452
and 2709) that corresponded to desthiobiotin-labeled Cys
active-site peptides. MS analysis of the p300 mixture resulted
in two different desthiobiotin-labeled peptides (m/z 2648 and
3182; Figure 2A, left), each of which contained a Cys unit
(Figure 2B). Fourier-transform MS unequivocally demon-
strated that the Cys-1621 was a site of interaction (Figure 2).
Confirming the importance of Cys-1621 in p300 acetyltrans-
ferase activity, C1621A resulted in a large rise in the Km values
of acetyl CoA (Table 1), which suggests that this Cys could be
Table 1: Comparison of kinetic parameters of p300 [wt] and C1621A
mutant.[a]
Received: June 7, 2007
Published online: September 4, 2007
Km (acetyl CoA)
Km (H4–15)
kcat (acetyl CoA)
p300
C1621A
6.4 mm
100 mm
30 mm
12 mm
0.21 sÀ1
0.24 sÀ1
Keywords: cofactors · enzymes · protein modifications ·
.
[a] For experimental details, see the Supporting Information; Km:
concentration for half-maximal rate, kcat: turnover number.
proteomics · transferases
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labeled on Cys-1653 near the C terminus (1666). Prior studies
have already implicated the Cterminus of the p300-HAT
domain as important.[4] These experiments illustrate the
utility of 2 in providing preliminary structural analysis of
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characterizing proteins present in cellular extracts. To exam-
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spiked with a small amount of p300. As shown in Figure 1C, a
range of proteins appear to be modified by 2 and the labeling
of several was selectively competed by actCoA. Among these,
p300 was readily identified and the modification efficiently
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endogenous GCN5-related acetyltransferase (GNAT)
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that was competed by excess actCoA (Figure 1D). In future
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identification of unknown CoA-binding proteins of interest in
signaling or metabolism. Moreover, we anticipate that the
sulfoxycarbonyl functionality may find other applications in
protein labeling.
Experimental Section
Details of the synthesis of the probe 2 are provided in the Supporting
Information.
Procedure for labeling recombinant proteins with sulfoxide 2:
Purified proteins (10 mL, 0.5 mgmLÀ1) were treated with 2 (25 mm;
865 mm stock in doubly distilled (dd) H2O) either with or without
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