(Received for publication, August 14, 1995)
From the
Kinetic as well as chemical modification studies have implicated
the presence of an active site arginine in choline acetyltransferase,
whose function is to stabilize coenzyme binding by interacting with the
3`-phosphate of the coenzyme A substrate. In order to identify this
residue seven conserved arginines in rat choline acetyltransferase were
converted to alanine by site-directed mutagenesis, and the properties
of these mutants were compared with the wild type enzyme. Substitution
of arginine 452 with alanine resulted in a 7-12-fold increase in
the K for both CoA and acetylcholine as
well as k
, with little change in the K
for dephospho-CoA. Product inhibition
studies showed choline to be a competitive inhibitor with respect to
acetylcholine, indicating R452A follows the same Theorell-Chance
kinetic mechanism as the wild type enzyme. Similar results were
obtained with R452Q and R452E, with the latter showing the largest
changes in kinetic parameters. These findings are consistent with
Arg-452 mutations increasing the rate constant, k
,
for dissociation of the coenzyme from the enzyme. Direct evidence that
arginine 452 is involved in coenzyme A binding was obtained by showing
a 5-10-fold decrease in affinity of the R452A mutant for coenzyme
A as determined by the ability to protect against phenylglyoxal
inactivation as well as thermal inactivation.
The enzyme choline acetyltransferase (ChAT, ()EC
2.3.1.6) catalyzes the transfer of the acyl group from acetyl-CoA to
choline resulting in the formation of the neurotransmitter
acetylcholine. Mechanistic studies on the enzyme suggest a concerted
reaction in contrast to similar enzymes that transfer acyl groups via
an acyl enzyme intermediate. The kinetic mechanism for the enzyme
approximates a Theorell-Chance mechanism(1) , although by using
isotope exchange at equilibrium a random component in the reaction has
been detected(2) . Chemical modification studies have
implicated histidine, cysteine, and arginine as active site residues.
Thus inactivation studies with dithiobis-4-nitro-2-carboxylate led to
the proposal that the enzyme contains an active site
cysteine(3, 4, 5, 6, 7) ;
however, modification of this residue by methylation showed it is not
essential for catalysis(8) . Similarly, an active site
histidine was implicated by inactivation studies with
diethylpyrocarbonate while an active site arginine was implicated by
inactivation studies with phenylglyoxal(7, 9) . It has
been suggested that the active site histidine serves as a general
acid/base catalyst(10) , while the active site arginine is
postulated to be involved in binding interactions with the 3`-phosphate
of the substrate CoA(9) .
With the availability of cDNA clones for ChAT from Drosophila melanogaster(11) , porcine spinal cord(12) , rat brain(13, 14) , mouse brain(14) , and Caenorhabditis elegans(15) it has now become possible to use site-directed mutagenesis to identify these active site residues and to study their function. We have previously used site-directed mutagenesis to analyze the functionality of three conserved histidines in Drosophila ChAT and have shown that one of these residues, His-426, is essential for catalysis(16) . We have now used a similar approach to search for the active site arginine thought to be involved in coenzyme A binding. Seven conserved arginines were individually changed to alanine and the resultant mutants characterized. The properties of mutant enzymes containing substitutions at arginine 452 are consistent with this arginine serving as an active site residue.
Previous studies have implicated an active site arginine residue to be involved in the binding of coenzyme A to the enzyme ChAT. Comparison of the amino acid sequences of ChAT from rat, Drosophila, and C. elegans, deduced from their respective cDNAs, revealed seven potential conserved arginines (Table 1). In order to determine which, if any, are involved in coenzyme binding we utilized a rat ChAT cDNA and alanine scanning in which each of these putative active site arginines was separately changed to an alanine by site-directed mutagenesis. Each cDNA was constructed in an expression vector containing an N-terminal hexahistidine fused to the ChAT protein. Previous studies (18) have established that this modification has no effect on the kinetic properties of the enzyme. Expression of the recombinant enzymes in E. coli, in all but one case, led to their facile purification by a two-step procedure involving metal affinity chromatography followed by dye binding chromatography as described under ``Experimental Procedures.'' Fig. 1shows representative preparations of the purified enzymes in which it can be seen that essentially homogeneous enzyme is obtained. In one case, the conversion of arginine 99 to alanine resulted in low levels of expression of this mutant, and difficulties were subsequently encountered in purifying this recombinant protein. Therefore, the properties of this mutant were analyzed in E. coli extracts rather than with purified enzyme.
Figure 1: SDS-PAGE analysis of purified recombinant ChAT mutants. 5 µg of the indicated enzyme was analyzed on an SDS-PAGE gel as described under ``Experimental Procedures.''
The kinetic properties of the
mutant enzymes were initially compared with the wild type enzyme in the
presence of 0.25 M NaCl, conditions previously shown to give
maximal activity(25) . The results of this kinetic analysis are
summarized in Table 2. Comparing the various alanine
substitutions, the most significant effect observed is with the mutant
R452A in which the K for CoA increases more than
12-fold, the K
for acetylcholine increases
8-fold, and k
increases
7-fold.
Smaller increases in the K
for CoA and
acetylcholine were observed with the mutants R453A, R458A, and R463A.
These studies were further extended by replacing arginine 452 with
glutamine, which is a near isosteric replacement, and also with
glutamate, which produces a reversal in charge at this position. The
kinetic properties of the R452Q mutant were essentially the same as the
R452A mutant, while the R452E mutant showed greater increases in the K
for CoA (54-fold), the K
for acetylcholine (60-fold), and k
(
15-fold) (Table 2). We also tested the effect of charge
reversal on the adjacent arginine, arginine 453, and the effect of
replacing both arginines 452 and 453 with glutamine. Charge reversal at
position 453 produced an enzyme form similar to R452A or R452Q, while
replacing both arginines produced an enzyme form in which the K
for CoA increased more than 170-fold, the K
for acetylcholine increased
70-fold, and k
increased
17-fold.
Since it has been
proposed that an active site arginine functions to interact with the
3`-phosphate of coenzyme A(9) , the effect of each mutation
with dephospho-CoA as substrate was examined, since no such interaction
should occur with this substrate. With the wild type enzyme
dephospho-CoA exhibits a K that is
10-fold
higher than coenzyme A (Table 2). In each of the mutants the K
for dephospho-CoA was similar to that obtained
with the wild type enzyme, this being particularly notable with R452E
and R452Q/R453Q in which the K
for dephospho-CoA
increased less than 4-fold as compared with changes in the K
for CoA of more than 50-fold for these mutants.
Thus at this initial level of analysis Arg-452 appears to be a likely
candidate as an active site residue, with arginines 453, 458, and 463
also being possible candidates.
It has been previously observed that
anions affect the kinetic parameters of the ChAT reaction(26) .
That is, anions act to increase V but at the
same time increase the K
for both
substrates(25) . It has been suggested that this effect can be
attributed, at least in part, to anions interfering with the binding of
the 3`-phosphate of coenzyme A to an active site arginine(9) .
Thus a similar kinetic analysis was conducted with the wild type enzyme
and the four candidate active site arginine mutants under conditions of
low ionic strength. The results of this analysis are shown in Table 3. In agreement with previous
studies(25, 26) , it can be seen that with the wild
type enzyme decreasing the anion concentration decreases the K
for CoA
12-fold but decreases the K
for choline less than 3-fold and lowers k
by approximately 2-fold. In contrast
decreasing salt has little effect on the K
for
dephospho-CoA, a finding consistent with the proposal that anions
compete for the interaction at the 3`-phosphate of coenzyme A with an
arginine.
The effects of decreased ionic strength are different with
the R452A mutant. The K for CoA is reduced only
3-fold by lowering the ionic strength of the assay, while there is
little change in the K
for acetylcholine and k
is essentially unchanged. As with the wild
type enzyme the K
for dephospho-CoA is barely
affected by changes in anion concentration. The other putative active
site arginine mutants all exhibit changes in their kinetic properties
that are similar to those observed with the wild type enzyme except
that k
remained unchanged. Again these results
are consistent with arginine 452 as the active site arginine
interacting with CoA.
In order to test for a change in kinetic
mechanism between the wild type enzyme and the R452A mutant we
determined the inhibition pattern with choline as a product inhibitor.
The ChAT reaction follows primarily a Theorell-Chance kinetic
mechanism, characterized by competitive inhibition between the inner
substrate pair choline and acetylcholine(25) . As shown in Fig. 2, choline acts as a competitive inhibitor with respect to
acetylcholine for both the wild type enzyme and for the R452A mutant, a
finding suggesting that both enzymes exhibit the same Theorell-Chance
kinetic mechanism. However, the K for choline was
greater for the mutant (1.1 mM) as compared with the wild type
enzyme (0.1 mM). Although not shown, competitive inhibition
between choline and acetylcholine was also observed with the most
severely affected mutant R452Q/R453Q with the K
for choline increased to 12 mM.
Figure 2:
Choline as a competitive inhibitor of wild
type ChAT and R452A. Choline inhibition was determined by the
fluorometric assay in the presence of 0.25 M NaCl, variable
acetylcholine, and a fixed level of CoA (2 K
). Data were plotted as
1/v versus 1/[AcCh]. K
was calculated from the equation for a competitive
inhibitor, v = V
(K
/[AcCh]{1 +
[I]/K
} + 1). Choline
inhibition of wild type enzyme:
, no added choline;
, 0.1
mM choline;
, 0.3 mM choline. Choline
inhibition of R452A mutant:
, no added choline;
, 1.0
mM choline;
, 3.0 mM choline.
We next analyzed the
ability of each of the alanine mutants to react with the
arginine-specific reagent phenylglyoxal. As shown in Fig. 3treatment of the wild type enzyme with 5 mM phenylglyoxal results in biphasic inactivation. There is a rapid
initial decline of activity, which is too fast to measure but which
results in a loss of 35% enzyme activity. This is followed by a
slower loss in enzyme activity, which plateaus at
90%
inactivation. A replot of the data as log (% activity remaining) versus time from 0.1 to 45 min is linear assuming an end point
of 90% inactivation. From this analysis a half-time of
11 min was
obtained. As shown in Fig. 3the slow phase of inactivation was
prevented by inclusion of acetyl-CoA in the inactivation reaction at a
concentration in the range of the K
for CoA.
Acetylcholine was without effect. Inclusion of acetyl-CoA at
concentrations greater than 50 times the K
for CoA
had no effect on the fast phase (data not shown). Conversion of
arginines 99, 312, and 453 to alanine had no significant effect on
phenylglyoxal inactivation. In each of these cases the same extent of
enzyme inactivation was observed in the rapid phase, and the half-time
for the slow phase varied only slightly from that observed with the
wild type enzyme (10, 12, and 10 min for alanine substitutions at
arginines 99, 312, and 453, respectively).
Figure 3:
Phenylglyoxal inactivation of wild type
and Arg Ala mutants of rat ChAT. Phenylglyoxal inactivation was
conducted as described under ``Experimental Procedures'' in
the presence of 5 mM phenylglyoxal and when added
acetylcholine or acetyl-CoA. In each case inactivation in the presence
of 5 mM phenylglyoxal is designated as the open square while the control incubation in the absence of phenylglyoxal is
given by the filled square. Upper left panel,
inactivation of wild type enzyme as noted and in the presence of 1.8
mM acetylcholine (
) or 0.5 µM acetyl-CoA
(
); upper right panel, inactivation of R250A; lower
left panel, inactivation of R452A as noted and in the presence of
0.5 µM acetyl-CoA (
) or 13.6 µM acetyl-CoA (
); lower right panel, inactivation of
R463A as noted and in the presence of 2.5 µM acetyl-CoA
(
).
Changing arginine 452 to
alanine resulted in a biphasic inactivation curve similar to the wild
type enzyme except that the secondary phase was faster (t
7 min) and inactivation went to
completion Fig. 3. Acetyl-CoA at the same concentration used to
protect the wild type enzyme from inactivation was ineffective with
this mutant; however, increasing the acetyl-CoA concentration to 13.6
µM was able to afford protection (Fig. 3). Although
not shown, acetylcholine at 15 mM had no effect on
phenylglyoxal inactivation.
Changing arginine 463 to Ala resulted in a considerably more rapid rate of inactivation by phenylglyoxal (Fig. 3). In this case the initial rapid and secondary phases of the reaction could not be distinguished. However, using 2.5 µM acetyl-CoA to protect against inactivation, the biphasic nature of the inactivation process became apparent (Fig. 3). Changing Arg-250 to Ala totally eliminated the secondary phase of inactivation by phenylglyoxal (Fig. 3).
In order to provide additional
evidence that Arg-452 is involved in CoA binding, we measured the
ability of coenzyme A to protect the enzyme against thermal
inactivation. The wild type enzyme is inactivated at 48 °C under
low ionic strength conditions with a t of 60 s.
As shown in Fig. 4, CoA afforded partial protection against
thermal inactivation exhibiting a K
value of
0.4 µM, a value similar to the kinetic K
of 0.25 µM listed in Table 2.
The R452A mutant was more thermolabile being inactivated at 48 °C
with a t
of
20 s. At 44 °C the t
was 35 s, and CoA also provided partial
protection against thermal inactivation; however, in this case the
estimated binding constant was shifted to
6 µM.
Figure 4: CoA protection of thermal inactivation of wild type ChAT and R452A. Left, wild type enzyme inactivated at 48 °C in the presence of the indicated concentration of CoA. Half-times were obtained from plots of log activity remaining versus time at each CoA concentration. Right, R452A inactivated at 44 °C in the presence of the indicated concentration of CoA. Half-times were obtained from plots of log activity remaining versus time at each CoA concentration.
Previous studies have shown that the enzyme choline
acetyltransferase is inactivated by arginine-specific reagents in a
reaction protected by the substrate acetyl-CoA(9) . This
observation, in conjunction with the results of kinetic studies which
showed that dephospho-coenzyme A was poorly bound by the
enzyme(6) , led to the proposal of an active site arginine that
interacts with the 3`-phosphate of coenzyme A. Since the results of
chemical modification experiments are often equivocal or ambiguous, we
utilized alanine scanning to systematically search for this active site
arginine by replacing seven conserved arginines with alanine. The
results of the kinetic analysis of these mutant enzymes are consistent
with arginine 452 being the likely candidate. The predicted properties
of such a mutant would include a decreased affinity for CoA with little
change in the affinity of the mutant for dephospho-CoA. In accordance
with this predicted behavior conversion of arginine 452 to alanine,
glutamine, or even glutamate caused a 12-50-fold increase in the K for CoA but less than a 3-fold change in the K
for dephospho-CoA. Thus in contrast to the wild
type enzyme, where the K
for CoA is
10-fold
lower than the K
for dephospho-CoA, the K
values for CoA and dephospho-CoA are nearly the
same in R452A and R452Q. With R452E dephospho-CoA becomes a better
substrate than CoA. The observation that the properties of R452A and
R452Q are quite similar rules out any complications that might have
been introduced as a result of a change in the side chain volume when
substituting the non-isosteric alanine for arginine. Thus this data is
consistent with arginine 452 interacting with the 3`-phosphate of
coenzyme A. The effects of changing arginine 452 by mutagenesis may be
blunted by the presence of an adjacent arginine in position 453.
Arginine 453 may also be directly involving in coenzyme binding or
alternatively may realign in the Arg-452 mutants so that it can
participate in coenzyme binding. This might explain why the most
dramatic effects are seen with the R452Q/R453Q double mutant in which
both arginines have been substituted.
At first glance it might seem
surprising that the K for both CoA and choline as
well as k
would be increased by mutations at
position 452. In the Theorell-Chance kinetic mechanism, which the
Arg-452 mutant appears to obey, the rate-limiting step in the reaction
is the release of product from the enzyme, k
in Fig. S1. The K
values for coenzyme A and
acetylcholine reflect their respective affinity in the steady state and
are defined as the ratio of k
/k
and k
/k
, respectively. The data
are consistent with mutations at Arg-452 decreasing the affinity of the
enzyme for CoA (or acetyl-CoA) by loss of the interaction with the
3`-phosphate. Kinetically this is manifested as an increase in the rate
constant k
(k
), which
represents dissociation of the coenzyme from the enzyme. An increase in k
would also be expected to result in an increase
in K
since this kinetic
constant is directly proportional to k
. With R452A
and R452Q there is a constant 7-12-fold increase in k
, K
, and K
, a finding consistent with this
proposal. The greater effects on K
and K
than on k
as seen in R452E and R452Q/R453Q may reflect other structural
changes in the enzyme or may be due to the presence of inactive enzyme
since the activity of these mutants was found to be rather labile. The
increase in kinetic constants seen in R453E could likely result from
ionic interactions between the introduced glutamate and the arginine at
position 452. This would result in a decreased interaction of arginine
452 with the coenzyme, making this mutant resemble an Arg-452 mutant.
Figure S1: Scheme 1. Kinetic scheme for the choline acetyltransferase reaction.
In low salt the K decreases
12-fold, while the K
for dephospho-CoA
changes less than 2-fold. Conversion of arginine 452 to alanine results
in a relatively small decrease in the K
for CoA
and eliminates the increase in V
. If as
postulated, k
is increased by anions as a result
of disrupting the interaction of an active site arginine with the
3`-phosphate of CoA, this effect would be expected to be eliminated
when arginine is converted to alanine. Thus, as predicted, the R452A
mutant, in contrast to the wild type enzyme, is refractory to salt
effects.
Further evidence that arginine 452 is involved in coenzyme
A binding is the demonstration that acetyl-CoA protection against
phenylglyoxal inactivation requires coenzyme concentrations
approximating the K for CoA for the R452A mutant
and is ineffective at the lower acetyl-CoA concentration that protects
the wild type enzyme. Similarly protection against heat inactivation
requires CoA concentrations in the range of its K
for the R452A mutant, while a considerably lower concentration
protects the wild type enzyme. It is interesting to note that the
residue which reacts with phenylglyoxal appears to be arginine 250.
Changing this residue to alanine has no effect on the kinetic
properties of the enzyme, indicating that its reaction with
phenylglyoxal results in either a conformational change or steric
hindrance. Modification of this residue does not result in a loss in
activity, supporting the conclusion that this is not a critical residue
for catalysis. Changing arginine 452 or 463 to alanine increased the
rate as well as the extent of inactivation by phenylglyoxal. This
indicates that these mutations permitted either an additional residue
to react with phenylglyoxal or resulted in a structural change such
that reaction with arginine 250 totally blocked activity.
Interestingly, conversion of three other arginines, Arg-453, -458, and -463, to alanine also affects the kinetics of the reaction, albeit to a considerably lesser extent. These arginines are clustered in the primary sequence. Thus it is possible that these residues are situated in the vicinity of or actually constitute part of the active site in three-dimensional space, participating in coenzyme A binding. It is worth noting that x-ray crystallographic studies have shown that there are multiple interactions such as electrostatic interactions, hydrophobic interactions, and hydrogen bonds between coenzyme A and the enzyme citrate synthase(27) . At least three arginines form salt bridges with the three charged phosphate moieties; Arg-46 and Arg-324 (through a water molecule) interact with the 5`-diphosphate while Arg-164 interacts with the 3`-phosphate in addition to possible hydrogen bonding interactions.
In summary alanine scanning of conserved arginines in the choline acetyltransferase reaction has provided evidence for arginine 452 being an active site residue that functions by interacting with the 3`-phosphate of the substrate CoA/acetyl-CoA. This arginine lies within a highly conserved region of the enzyme, whose sequence varies little among such divergent species as mammals, Drosophila, and the nematode, C. elegans. Direct evidence for this proposal must await the determination of the three-dimensional structure of the enzyme.