(Received for publication, August 10, 1995)
From the
Escherichia coli galactoside acetyltransferase (GAT) is
a member of a large family of acetyltransferases that O-acetylate dissimilar substrates but share limited sequence
homology. Steady-state kinetic analysis of overexpressed GAT
demonstrated that it accepted a range of substrates, including
glucosides and lactosides which were acetylated at rates comparable to
galactosides. GAT was shown to be a trimeric acetyltransferase by
cross-linking with dimethyl suberimidate. Fluorometric analysis of
coenzyme A binding showed that there is a fluorescence quench
associated with acetyl-CoA binding whereas CoA has no effect. This
difference was exploited to measure dissociation rates for both CoA and
acetyl-CoA by stopped-flow fluorometry. The rate of dissociation of CoA
(2500 s) is at least 170-fold faster than k
for any substrate tested. The fluorescence
response to acetyl-CoA binding is entirely due to Trp-139 since
replacement by phenylalanine completely abolished the fluorescence
quench. Treatment of GAT by [
C]iodoacetamide
resulted in complete inactivation of the enzyme and the incorporation
of label into histidyl and cysteinyl residues to approximately equal
extents. Following replacement of His-115 by alanine, label was
incorporated solely into cysteinyl residues. Furthermore, the
substitution results in an 1800-fold decrease in k
suggesting that His-115 has an important catalytic role in GAT.
Galactoside acetyltransferase (EC 2.3.1.18; GAT) ()transfers an acetyl group from acetyl-CoA to the
6-hydroxyl of certain galactopyranosides(1) . GAT is encoded by
the lacA gene of the lac operon of Escherichia coli,
and consequently lactopyranosides induce expression of lacA coordinately with the lacZ and lacY genes which
encode
-galactosidase and lactose permease,
respectively(2) . The latter two enzymes have clearly defined
roles in the catabolism of lactose whereas GAT does not; its function
in microbial metabolism remains ill-defined since lacA plus
and minus strains of E. coli are equally viable under
laboratory conditions(3) . Andrews and Lin (4) proposed
that the lacA gene product might have a role in
detoxification, by facilitating the elimination of non-metabolizable
lactopyranosides from the cell since acetylated lactopyranosides are
not taken up by cells (3) .
E. coli GAT has been studied extensively by Zabin and co-workers(5, 6, 7, 8) . Both the DNA sequence of the lacA gene (5) and the amino acid sequence of GAT (6) have been determined independently. Although GAT was reported to be dimeric(7) , the subsequent determination of the exact subunit molecular mass (22,700 Da (6) ) from sequencing together with a value of 65,300 Da from ultracentrifugation studies for the native molecular mass (8) suggests that GAT might be a trimer. Steady-state kinetic studies with IPTG as acetyl acceptor indicated that GAT catalyzed acetyl transfer by a compulsory order ternary complex mechanism with acetyl-CoA as the leading substrate(9) .
GAT is a member of a large family of acetyltransferase enzymes that share regions of amino acid sequence homology. Downie (10) reported extensive homology throughout the amino acid sequence of GAT and of the nodL gene product of Rhizobium leguminosarum, a lipooligosaccharide acetyltransferase. Although the precise nature of the substrate for lipooligosaccharide acetyltransferase is unclear, like GAT it acetylates the 6-hydroxyl group of a galactopyranoside ring (11) . More limited areas of homology have been found between the C-terminal regions of the amino acid sequences of GAT, serine acetyltransferases, and a class of enzymes that may conveniently be described as xenobiotic acetyltransferases(10, 12) . Since enzymes of the xenobiotic acetyltransferase class acetylate antibiotics such as chloramphenicol as well as virginamycin-like compounds(12, 13, 14) , the evident dissimilarity of the acetyl acceptors for this class of acetyltransferases suggests that the region of homology in their amino acid sequences could represent a structural feature required for binding coenzyme A.
GAT shows no primary structure homology to a
particularly well defined example of an acetyltransferase enzyme, type
III chloramphenicol acetyltransferase (CAT), for which a
three-dimensional structure is available(15, 16) .
CAT
is a homotrimer (3
25 kDa) with a kinetic
mechanism that involves the formation of a ternary complex via a random
order of addition of substrates(17) . The catalytic mechanism
of CAT
has been studied in some detail. The N
of the imidazole of His-195, conserved in all members of the CAT
family, acts as a general base, abstracting a proton from the hydroxyl
group of chloramphenicol, thereby promoting nucleophilic attack by the
oxygen at the carbonyl of the thioester of acetyl-CoA. The mechanism
proceeds via a tetrahedral intermediate wherein the resulting oxyanion
is stabilized by hydrogen bonding to the hydroxyl of Ser-148, another
conserved residue. Substitution of Ser-148 and His-195 by Ala in
CAT
results in 53-fold and 9
10
-fold
decreases in k
,
respectively(18, 19) . Another class of
acyltransferases that may be distinct from either the CAT or the GAT
family includes choline acetyltransferase and carnitine
palmitoyltransferase, both of which have also been reported to have
catalytic histidyl residues(20, 21) .
In the present study on GAT we show that GAT is indeed a trimeric acetyltransferase, report the results of steady-state and pre-steady-state kinetic studies of the enzyme, and examine the fluorescence response of GAT to coenzyme binding. We also present evidence from site-directed mutagenesis and chemical modification studies that suggests that GAT contains a catalytically important histidyl residue.
Oligonucleotide-directed mismatch mutagenesis was
performed using the deoxyuridine selection protocol with the dut
ung E. coli strain RZ1032(23) . The oligonucleotides
(mismatches underlined) used were: 5`-GGTGTACAGGGGCTCCCGTAACG (His-115
Ala), 5`-GACTTCCGATA(T/A)AGACGTTATTG (Trp-139
Phe or
Tyr), and 5`-TTGTGACGATAGCACCCGCGCCA (Ser-162
Ala). The presence
of the desired nucleotide substitution and the absence of second site
mutations were confirmed by sequence analysis. Mutant lacA determinants were overexpressed in E. coli JM101 or E. coli X90
LacA46 in the case of H115A GAT
following transfer to pKK223-3 as described above.
[C]Iodoacetamide was diluted with unlabeled
reagent to give a final specific activity of 168 MBq/mmol. Wild-type
GAT was incubated in TSE buffer in the presence of 50 mM [
C]iodoacetamide. Samples were withdrawn at
intervals and the extent of covalent incorporation was monitored by
filter binding as described by Kleanthous et al.(30) .
In preparative analyses, wild-type GAT and the H115A variant (100 nmol)
were each treated with 50 mM [
C]iodoacetamide. After 4 h the reaction
was quenched by adding 2-mercaptoethanol (final concentration 0.1 M) and a sample of the treated protein in each case was
counted to establish the stoichiometry of modification. The remainder
was subjected to acid hydrolysis, and the products were resolved by
thin layer electrophoresis (Polygram SilG silica)(30) .
Following staining with fluorescamine, the plate was cut into strips
and analyzed by scintillation counting to identify modified amino
acids.
Generally, the major
effect of altering either the acetyl donor or the acceptor is expressed
in changes in k. Altering the structure of the
acetyl acceptor has little effect on the K
value
for either substrate, with the exception of the K
for acetyl-CoA with PNP
Lac as acetyl acceptor. However, the
changes in k
suggest that PNP
Gal is the
best substrate, followed by ONP
Gal, whereas PNP
Gal,
PNP
Glc, and PNP
Lac are all relatively poor substrates.
Moreover, the presence of the nitro group on the phenyl ring makes
little difference since phenyl-
Gal approaches PNP
Gal in
efficacy as an acetyl acceptor. Furthermore, since p-nitrophenol-
-D-N-acetylglucopyranoside
is acetylated approximately 20-fold more slowly than PNP
Glu, GAT
appears to prefer sugars without N-acetyl groups. The major
effect of increasing the chain length of the acetyl donor is a decrease
in the value of k
. Although propionyl-CoA is
almost as effective as acetyl-CoA as an acyl donor (k
decreased only 2.5-fold), butyryl-CoA is a very poor substrate; k
is decreased 270-fold and K
is increased 2-fold compared to acetyl-CoA.
Musso and Zabin (9) proposed that GAT follows an ordered sequential mechanism
with acetyl-CoA as the leading substrate. If such a mechanism applies,
it should be possible to extract K values for
acetyl-CoA from the results of steady-state kinetic analysis, and show
that such values for K
do not depend on the
structure of the acetyl acceptor. The data for acetyl-CoA, shown in Table 1, confirms that the nature of the acetyl acceptor has
little effect on the kinetically derived K
values,
as expected for the proposed mechanism.
Figure 1: Intrinsic protein fluorescence response to the binding of acetyl-CoA and CoA. Fluorescence emission spectra are: 1, wild-type GAT; 2, wild-type GAT with 200 µM acetyl-CoA; 3, wild-type GAT with 200 µM CoA; 4, W139F GAT; 5, W139F GAT with 200 µM acetyl-CoA; and 6, W139F GAT with 200 µM CoA. The excitation wavelength was 297 nm.
The results observed with unesterified CoA as the ligand are somewhat unexpected in that, despite the marked quench of fluorescence intensity on binding acetyl-CoA, GAT shows virtually no fluorescence response on association with CoA (Fig. 1).
The fluorescence quench induced by
acetyl-CoA binding allowed the direct determination of the dissociation
constant for the GATacetyl-CoA binary complex by fluorometric
titration. The K
value of 20.8 µM obtained by this technique is significantly lower than the values
estimated from steady-state kinetics. Binding of the substrate
analogue, ethyl-CoA, also results in a fluorescence quench of the same
magnitude as that produced by acetyl-CoA, however, the K
for ethyl-CoA is approximately 300 µM.
A sample of 4 µM GAT was
preincubated with 150 µM acetyl-CoA (syringe
concentrations) and mixed with 400 µM CoA in the
stopped-flow apparatus. The observed increase in fluorescence (due to
the displacement of acetyl-CoA) was small (2%) and, when fitted to
a single exponential expression, occurred at a rate in excess of 600
s
at 25 °C. In the reciprocal experiment, the
dissociation rate constant for CoA was found to be too fast to measure
at 25 °C in that no fluorescence change was observed.
In view of
this, together with the uncertainty inherent in monitoring small
amplitude changes at very fast rates for acetyl-CoA dissociation, the
procedure was repeated at a number of lower temperatures. On plotting
these data as ln(k) versus temperature
(results not shown), the linear fit was extrapolated to estimate the
dissociation rate at 25 °C. For acetyl-CoA this rate was found to
be approximately 720 s
, and a much faster rate of
the order of 2500 s
for CoA.
From fluorometric
titrations, the K of acetyl-CoA was found to be
20.8 µM and, knowing k
720 s
, k
may be calculated to
be 3.5
10
M
s
. Clearly,
neither the association of acetyl-CoA nor the dissociation of CoA
during product release are rate-limiting in the transacetylation
reactions studied here.
The emission spectra in Fig. 1demonstrate the loss of intensity (46%) due to the
replacement of Trp-139 by phenylalanine. It is clear that the two
tryptophan residues within each GAT monomer contribute equally to the
observed fluorescence of the wild-type native trimer. The simple
additive nature of these fluorescence contributions indicates that
homogeneous radiation-less transfer between them does not have a
significant effect on their fluorescence properties when excited at 297
nm, near the red edge of the absorption spectrum for tryptophan.
The
position of the maximum emission wavelength varies slightly between the
GAT variants, reflecting subtle differences in the nature of the local
environment of each tryptophan (Fig. 1). The spectrum of W139F
GAT, retaining only Trp-63, shows a red shift in the emission peak
( = 341 nm), compared to the spectrum of
wild-type GAT (
= 337 nm); indicative of
either a relatively hydrophobic local environment for Trp-139 or
greater solvent accessibility for Trp-63.
W139F GAT retains little or no fluorescence response to acetyl-CoA (or CoA) association (Fig. 1). Hence, it is almost certain that Trp-139 is entirely responsible for the tryptophan-related fluorescence response of wild-type GAT to the binding of acetyl-CoA.
Figure 2:
a,
inactivation of GAT with iodoacetamide. GAT was treated with
iodoacetamide as described under ``Experimental Procedures.''
, 50 mM iodoacetamide;
, 50 mM iodoacetamide with 1 mM ethyl-CoA, and +, 50
mM iodoacetamide with 40 mM PNP
Gal. b,
stoichiometry of iodoacetamide inactivation. GAT was treated with
[
C]iodoacetamide as described under
``Experimental Procedures.'' At intervals, samples were
removed and the fraction of the initial activity remaining and the
extent of incorporation of
C label into the protein were
determined. c, high voltage electrophoresis of acid
hydrolysates of wild-type and H115A GAT modified by
[
C]iodoacetamide. Hydroysates were subjected to
electrophoresis at pH 6.5 on a silica plate. The plate was stained with
fluorescamine, the tracks were cut into strips, and the radioactivity
was located by liquid scintillation counting of the strips. The
positions of carboxymethylated amino acid standard compounds after
electrophoresis are indicated: A, 1-carboxymethylhistidine; B, 3-carboxymethylhistidine; C,
1,3-dicarboxymethylhistidine; and D,
carboxymethylcysteine.
The
stoichiometry of inactivation was examined by treatment of GAT with
[C]iodoacetamide (Fig. 2b).
Clearly, incorporation of label does not directly reflect inhibition of
GAT activity suggesting that iodoacetamide modifies more than one
residue in GAT. In fact, after 4 h, approximately 2 nmol of
C label were incorporated per nanomole of GAT monomers. In
order to determine the type of residue modified by the reagent,
[
C]iodoacetamide treatment of both wild-type and
H115A GAT was followed by acid hydrolysis and the resulting labeled
amino acids were separated by thin-layer electrophoresis(30) .
In each case the stoichiometry of inactivation was similar,
approximately 2 nmol of label incorporated per nmol of enzyme monomer.
However, the pattern of residues modified in the wild-type and
substituted enzymes were different (Fig. 2c). In
wild-type GAT, carboxymethylcysteine and 3-carboxymethylhistidine were
found in approximately equal amounts (37.8 and 49.3% of the total
counts recovered, respectively) indicating that iodoacetamide modifies
both cysteinyl and histidyl residues in the wild-type protein. In
contrast, in H115A GAT carboxymethylcysteine is the major product
(84.8% of total counts recovered), suggesting that His-115 is the
histidyl residue modified by iodoacetamide in wild-type GAT.
Cross-linking GAT with dimethyl suberimidate has confirmed
the suggestion from earlier M determinations that
GAT is a trimeric acetyltransferase. There is also evidence that a
xenobiotic acetyltransferase enzyme that acetylates chloramphenicol in Agrobacterium tumefaciens is a trimer. (
)Other
members of the acetyltransferase family to which GAT belongs may also
prove to be trimeric.
It is clear from steady-state kinetic analysis that GAT has a broad substrate specificity since it will acetylate galactosides, glucosides, and lactosides. Such a broad specificity supports the proposal (4) that GAT may act as a detoxifying enzyme, acetylating non-metabolizable sugars to prevent their re-entry into the cell.
The kinetic results support the mechanism postulated
by Musso and Zabin (9) . Certainly, acetyl-CoA binds to the
free enzyme and the association rate constant for acetyl-CoA and the
dissociation rate constant for CoA are much greater than k. The rate-determining step is therefore likely
to be either the loss of acetylated acceptor from the binary complex or
the interconversion of the substrate and the product ternary complexes.
The fluorescence response to acetyl-CoA binding has been shown to be
entirely due to Trp-139. Moreover, this tryptophanyl residue is in the
C-terminal portion of GAT that is homologous to other acetyltransferase
sequences. In fact, Trp-139 is found in other acetyltransferases
including the nodL product (LAT) as well as enzymes of the
xenobiotic acetyltransferase class, but not in serine acetyltransferase
where it is replaced by a methionyl residue. Since the K value for acetyl-CoA for W139F GAT is similar to that of
wild-type, it is unlikely that acetyl-CoA binding has been impaired by
this substitution. The intrinsic fluorescence of GAT is quenched by
acetyl-CoA but not CoA suggesting that Trp-139 may be close to the
binding site of the acetyl group of the thioester and consequently to
the active site of GAT. The fact that the Trp-139
Phe
substitution results in a substantial decrease in k
supports this hypothesis, but the observed differentiation
between acetyl-CoA and CoA may be due to another and more subtle
effect. In the better characterized example of CAT
, the
intrinsic fluorescence of Trp-152 is enhanced by association of
acetyl-CoA but not CoA(27) , although Trp-152 is in fact 7
Å from the thiol of CoA in the structure of the
CAT
CoA binary complex and would be expected, from
structural considerations alone, to respond equally well to CoA or
acetyl-CoA binding.
It is clear that Ser-162 appears to have no
catalytic function despite its conservation throughout most of the
acetyltransferase family. In contrast, His-115 seems certain to be
important for catalysis, although it is conserved only in the alignment
between GAT and the nodL product. The 1800-fold decrease in k in H115A GAT is a modest one compared to the 9
10
-fold decrease in k
observed for CAT
upon replacement of His-195 by
Ala(19) . Although it is possible that the function of His-115
may not be strictly analogous to that of His-195 in CAT
,
nonetheless it is clearly of critical importance to the catalytic
activity of GAT.
Chemical modification with iodoacetamide results in inactivation of GAT. The modification of histidyl and cysteinyl residues alike in wild-type GAT, but only of cysteinyl residues in H115A GAT, suggests that iodoacetamide modifies His-115 and one or both of the two cysteinyl residues of GAT. The fact that the stoichiometry of inactivation of H115A GAT is 2:1 suggests that both of the cysteinyl residues of GAT are fully modified in this variant, whereas in wild-type GAT either only one cysteine is modified or both cysteinyl residues are partially modified. It is unlikely that the cysteinyl residues of GAT have a catalytic function since, unlike His-115, neither are conserved in the sequence alignment between GAT and the nodL product (LAT).
Our interest in extending the work of others on the lacA gene product has arisen in the main from a desire to rationalize structure-function relationships for a large and growing apparent ``superfamily'' of microbial acetyltransferases that may, in time, be separated into subgroups by various criteria. Although in some respects the prototype of the superfamily, CAT shows no sequence homologies with the enzymes mentioned here and with GAT in particular. What does stand out thus far is the likelihood that GAT is, like CAT, a trimeric enzyme with a similar kinetic mechanism and a critical histidyl residue. Studies in progress on the nodL product (LAT), on serine acetyltransferase, and on members of the curious ``xenobiotic acetyltransferase'' class of heterogeneous acetyltransferases should help to clarify the co-evolution of structure and function within the proposed superfamily.