(Received for publication, December 8, 1994; and in revised form, June 8, 1995)
From the
In the past, purification of choline acetyltransferase (ChAT, EC
2.3.1.6), the enzyme responsible for the biosynthesis of the
neurotransmitter acetylcholine, has yielded fragmented species of the
enzyme. The nature and possible function of these forms of ChAT are not
well understood. Using a bacterial expression system, recombinant rat
ChAT in its active form has been purified to homogeneity. The purified
enzyme was found to be activated to >25-fold when assayed at low
ionic strength and >5-fold when assayed at high ionic strength by
limited proteolysis with either trypsin or chymotrypsin, but not with
proteinase K. The activated ChAT shows an increased K for both substrates, diminished
sensitivity to salt activation and a pH optimum that is shifted
approximately 1 pH unit. On a denaturing SDS-polyacrylamide gel, the
activated ChAT is composed of three to four polypeptides; however, it
migrates as an intact 68-kDa protein species on gel filtration. In
order to delineate the site of cleavage by proteolysis, the newly
generated fragments have been subjected to N-terminal sequencing. By
comparing cleavage sites between trypsin and chymotrypsin, the putative
activation sites were identified.
Choline acetyltransferase (ChAT, ()EC 2.3.1.6)
catalyzes the reversible transfer of an acetyl group between acetyl
coenzyme A and choline, resulting in the synthesis of the
neurotransmitter acetylcholine(1) . Since its discovery more
than 50 years ago(2) , ChAT has been extensively studied, but
little is known about structure/function relationships for this
molecule as a catalyst. Initially, the kinetic mechanism catalyzed by
this enzyme was postulated as an ordered bireactant or ping-pong
mechanism(3) . Now it is generally accepted that this is
predominantly an ordered sequential Theorell-Chance mechanism with
acetyl coenzyme A as the leading substrate, although a minor component
of a random sequential pathway does occur(4) . The reaction is
thought to proceed via general base catalysis by an imidazole residue
enhancing the nucleophilicity of the hydroxy group of choline to attack
the thioester bond in the forward reaction(5) . The presence of
an active site histidine was first postulated by White and
Cavallito(6) . Recently, this active site histidine has been
identified by site-directed mutagenesis as His-426 in Drosophila ChAT(7) .
Over the years multiple forms of choline acetyltransferase, which vary in charge and size, were observed using either column chromatography or electrophoretic methods(8, 9, 10) . However, the nature and physiological function of these variants remain unclear. Multiple charge forms of ChAT have been observed in all sources from which ChAT has been purified(8, 9, 10) . They do not represent distinct gene isoforms since the ChAT gene is a single copy in the genome(11) , nor do they represent alternative splicing products since a single transcript has the capacity to generate them(12) . Instead, multiple charge forms have been attributed to the possible association with other proteins(13) , to deamidation(14) , or to phosphorylation(15) . On the other hand, the size variants of ChAT may simply reflect artifacts generated during purification since they appeared to be somewhat variable depending on which purification scheme was used. For example, in the case of bovine brain ChAT, molecular sizes of 100-120 kDa(6, 8) , 72-76 kDa(13) , 65-70 kDa (16, 17) and six subunits of 14.7 kDa (18) have all been reported. Hersh et al.(19) attempted to address the nature of size and charge heterogeneity of ChAT from bovine brain and found that the 68-kDa form can be converted to a 63-kDa form by exogenously added Staphylococcus aureas protease, but not by autolysis in the extracts. However, only a 73-kDa form was observed when purification was carried out in the presence of a variety of proteolytic inhibitors(19) . Recently, Benecke et al.(20) reported the presence of 45- and 30-kDa forms, but not the intact 68-kDa form in partial purified ChAT samples. A 68-kDa form was, however, obtained in immunoaffinity-purified ChAT preparations(20) , suggesting that multiple sizes of ChAT were caused by proteolysis. In the case of Drosophila, cleavage of ChAT from 68 to 57 kDa and 13 kDa occurred with the wild-type enzyme, but not with two temperature-sensitive mutants(21) , indicating that proteolytic processing may be important for ChAT function. Thus, these studies clearly demonstrated the presence of proteolysis of ChAT, although the consequence and/or effects on catalysis were not investigated.
In order to address the nature of different size variants of ChAT and to better understand the process and effects proteolysis has on ChAT activity, limited proteolysis was carried out with known proteinases. In this report, we describe the activation of recombinant rat ChAT by limited proteolysis and the characterization of proteolytically activated enzyme.
The fluorometric assay followed the reverse reaction in which acetyl-CoA formation was measured in a coupled assay using citrate synthase and malate dehydrogenase. Reaction mixtures of 0.4 ml contained 50 mM sodium phosphate buffer, pH 7.4, 100 mM sodium chloride, 0.25 mM NAD, 0.5 mML-malate, 0.1 mMDL-dithiothreitol, 1.0 unit of pig heart citrate synthase, 5 units of pig heart malate dehydrogenase, and 10 mM acetylcholine chloride and 200 µM CoA. The reaction was initiated by the addition of acetylcholine chloride, and NADH formation was monitored over a 3-5 min time period using a Ratio-2 fluorometer equipped with a strip chart recorder. An excitation wavelength of 340 nm and an emission wavelength of 460 nm were used.
Figure 1:
Effect of trypsin
treatment on the activity of recombinant rat choline acetyltransferase.
Trypsin digestion () was carried out at 22 °C in 10 mM sodium phosphate, pH 7.6, with 1 mg/ml of ChAT and 1 µg of
trypsin in a reaction volume of 10 µl. As a control (
), enzyme
was incubated under the same conditions in the absence of trypsin. The
reaction was stop by the addition of 2 mg of soybean trypsin
inhibitor/mg of trypsin, and enzymatic activity was determined by the
standard fluorometric assay after a 200-fold dilution of the reaction
mix.
Figure 2: Gel filtration of rat recombinant choline acetyltransferase before and after incubation with trypsin. Opencircles are data for native choline acetyltransferase, and closedcircles are data for trypsin-treated choline acetyltransferase chromatographed on a Superdex 75 column at flow rate of 0.5 ml/min using 10 mM sodium phosphate buffer, pH 7.6, containing 100 mM sodium chloride.
Figure 3: SDS-PAGE analysis of recombinant choline acetyltransferase before and after limited proteolysis with trypsin. Native recombinant rat ChAT (5 µg; lane2) and trypsin-activated ChAT (5 µg; lane3) were analyzed on a 10% SDS-PAGE. Proteins were visualized by silver staining. The resultant tryptic peptides are labeled Ta, Tb, and Tc1, Tc2 and Tc3 for reference. Lane1, prestained molecular size markers. The arrows indicate, from top to bottom, phosphorylase b (107 kDa), bovine serum albumin (80 kDa), ovalbumin (45 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (26 kDa), and lysozyme (18 kDa).
Figure 4: The effect of sodium chloride concentration on enzyme activities of native and proteinase-activated choline acetyltransferase. ChAT activity was measured in the presence of increasing concentrations of sodium chloride at saturating substrate concentrations. Reactions were conducted at 37 °C for 10 min as described under ``Experimental Procedures'': opencircles represent activity of the native choline acetyltransferase, and closedcircles are those for choline acetyltransferase activated with trypsin.
The pH optima of both native and activated enzyme were examined at both low and high ionic strength (Fig. 5). It can be seen that the pH optimum of native ChAT is between pH 6.5 and 7 at the low salt condition, while that of the activated ChAT under the same conditions has been shifted to a value between pH 7.5 and 8. The pH optimum for both the native and trypsin-activated ChAT at high salt are both between pH 7.5 and 8.
Figure 5: Effects of pH on the activity of the native and activated choline acetyltransferase. Enzyme activity of native ChAT in low salt buffer (opencircles) and high salt buffer (opentriangles) and that of ChAT activated with trypsin in low salt buffer (closedcircles) and high salt buffer (closedtriangles). Assays were performed at the indicated pH values at 37 °C for 10 min as described under ``Experimental Procedures.''
Figure 6: SDS-PAGE analysis of recombinant choline acetyltransferase before and after limited proteolysis with chymotrypsin. Native recombinant rat ChAT (5 µg; lane2) and chymotrypsin-activated ChAT (5 µg; lane3) analyzed on a 10% SDS-PAGE. Proteins were visualized by silver staining. Lane1, prestained molecular size markers as in Fig. 3.
Figure 7: Schematic representation of the structures of proteolyzed ChAT. The N termini of newly generated ChAT fragments from either chymotrypsin (A) or trypsin (B) cleavage were determined by amino acid sequencing (see Table 4). Cleavage sites are indicated by the designation of the N-terminal amino acid found in each proteolytic fragment, which are labeled as in Table 4with their sizes indicated by dotted lines.
Recombinant rat ChAT after trypsin treatment exhibits altered
kinetic properties when compared with the native enzyme. The K values for both substrates increase
significantly, as does V
. The observed
5
6-fold increase in V
, at a physiological
ionic strength, is a minimal estimate since it has been shown with
endopeptidase Arg-C that cleavage leading to decreased activity can
also occur. Previous studies from this laboratory indicated that the
rate-limiting step in the acetylation reaction is primarily the
dissociation of the CoA product(30) . Therefore, activation may
represent a structural alteration from which the rate of CoA/acetyl-CoA
dissociation from the enzyme is increased. Based on its Theorell-Chance
mechanism, the K
for CoA is k
/k
, and the K
for ACh is k
/k
, while k
is defined as k
(see Fig. S1).
Figure S1: Scheme 1.
The activated enzyme exhibits an approximate
5-10-fold increase in the K value
for both of its substrates and a 5-fold increase in the turnover number k
, consistent with the hypothesis that limited
proteolysis causes an increase in the coenzyme A dissociation rate
constant, k
. The mechanism by which salt increases
choline acetyltransferase activity has also been proposed to be due to
an increase in the coenzyme A dissociation rate(30) .
There
are some interesting similarities between salt effects and proteolytic
activation of choline acetyltransferase. The pH activity profile of the
activated ChAT is very similar to that of the native enzyme in high
salt. High salt did not enhance the activity of proteolytically
activated ChAT under the same condition in which salt increased the
activity of native ChAT. Although high salt does not increase kto the same extent proteolysis does, it
seems feasible to suggest that the catalytic residues in
proteinase-activated ChAT may experience a similar structural change to
those of the native enzyme in high salt. Thus, it seems reasonable to
postulate that native choline acetyltransferase is normally in a
restrained conformation such that maximal catalytic turnover cannot be
achieved and that in the presence of high ionic strength buffer or
``fixed ion pairs'' generated by proteolysis, the
conformation of the enzyme undergoes alteration such that the
``optimal'' catalytic capacity is now able to be manifested.
The failure to ``super-activate'' trypsin-activated ChAT by
chymotrypsin or vise versa appears to agree with the proposed model.
N-terminal sequence analysis of ChAT activated either by trypsin or
chymotrypsin indicates that amino acids 14-18, 150-153,
360-363, and 480-481 are localized on the surface
accessible to proteinases. Not all cleavages of these bonds are
required for activation. For example, digestion of the 480-481
bond appeared unique to trypsin-activated ChAT, suggesting that it is
not a trigger for activation. This was proven to be true by mutagenesis
in which this trypsin recognition site was changed and trypsin
treatment still activated the mutant enzyme. ()The cleavage
around amino acids 150-153, although present in both trypsin and
chymotrypsin-activated ChAT, cannot be responsible for activation.
Cleavage of amide bonds around amino acids 150-153 leads to the
disappearance of the 31- or 33-kDa bands, which does not result in any
further enhancement of ChAT activity. Thus it appears that the cleavage
of either amino acids 14-18 and/or 360-363 are required for
activation.
The activation of ChAT by proteolysis was also observed
with mammalian ChAT purified from brain sources, and
therefore is not a result of expression of the recombinant protein. In
general, limited proteolysis leading to enzymatic activation can be an
important regulatory event in many physiological processes. For
example, zymogen activation is a controlled process in terms of
temporal and spatial regulation where large amounts of enzyme protein
can be initially synthesized and stored in its inactive state and
enormous activity can be generated by proteolytic activation in a short
period of time or upon release to the location of action. Limited
proteolysis has been shown to cause rearrangement of the conformation
of the zymogen. This phenomenon is generally seen where regulation at
the transcription level is too slow, and reversible mechanisms such as
group transfers are not well suited to create a permanent change in the
molecular environment. Choline acetyltransferase is synthesized in an
active state and it is not known at this time if an in vivo activation mechanism occurs at the site of physiological action by
limited proteolysis. However, it is worth noting that in mature sperm,
choline acetyltransferase is located in the middle ring of the acrosome
followed immediately in cellular space by acrosin, a trypsin-like
serine proteinase(31) . At lease three cholinergic markers have
been reported to exist in sperm, including the nicotinic receptor
acetylcholinesterase and choline acetyltransferase, and these
cholinergic components are believed to be involved in sperm maturation
and motility(32) . It seems possible that choline
acetyltransferase could be activated to increase the rate of
biosynthesis of ACh by acrosin. It is of interest to note that Cozzari
and Hartman observed both activator(s) and inhibitor(s) of mammalian
ChAT(13) . These factors could represent proteinases.
In summary, we have reported a novel proteolytic activation phenomenon for choline acetyltransferase and have addressed the activation mechanism by both kinetic and structural analysis.