(Received for publication, September 8, 1994; and in revised form, November 23, 1994)
From the
The amidotransferase or glutaminase (GLNase) domain of mammalian
carbamyl phosphate synthetase (CPSase), part of the 243-kDa CAD
polypeptide, consists of a carboxyl half that is homologous to all
trpG-type amidotransferases and an amino half unique to the carbamyl
phosphate synthetases. The two halves of the mammalian GLNase domain
have been cloned separately, expressed in Escherichia coli,
and purified. The 21-kDa carboxyl half, the catalytic subdomain, is
extraordinarily active. The k is 347-fold higher
and the K
is 40-fold lower than the
complete GLNase domain. Unlike the GLNase domain, the catalytic
subdomain does not form a stable hybrid complex with the E. coli CPSase synthetase subunit. Nevertheless, titration of the
synthetase subunit with the catalytic subdomain partially restores
glutamine-dependent CPSase activity. The 19-kDa amino half, the
interaction subdomain, binds tightly to the E. coli CPSase
large subunit. Thus, the GLNase domain consists of two subdomains which
can autonomously fold and function. The catalytic subdomain weakly
interacts with the synthetase domain and has all of the residues
necessary for catalysis. The interaction subdomain is required for
complex formation and also attenuates the intrinsically high activity
of the catalytic subdomain and, thus, may be a key element of the
interdomain functional linkage.
Amidotransferases catalyze glutamine hydrolysis to supply ammonia for de novo biosynthesis of purines, pyrimidines, glucosamine, folate, nicotinamide, and several amino acids(1, 2) . There are two classes of amidotransferase, trpG and purF types(2) , which have dissimilar amino acid sequences but a common catalytic mechanism(3) . In both types of enzymes, the amidotransferase activity is usually associated with a separate subunit or domain of the biosynthetic complex. The trpG amidotransferase domains represent a homologous family of proteins with a molecular mass of 20 kDa and a sequence that includes three nearly invariant regions (regions I-III) shown to be important for the function of the molecule(2, 3) .
Carbamyl phosphate synthetase
(CPSase, ()EC 6.3.5.5), a trpG type amidotransferase, (
)catalyzes the formation of carbamyl phosphate from
bicarbonate, 2 mol of ATP, and glutamine(4) . Escherichia
coli carbamyl phosphate synthetase (5) consists of a small
(42-kDa) subunit which carries glutaminase activity, and a large
(118-kDa) subunit which catalyzes ammonia-dependent carbamyl phosphate
synthesis. The structural organization of the enzyme is quite different
in mammals. De novo pyrimidine biosynthesis is initiated by
CAD(6, 7, 8) , a large multifunctional
protein which catalyzes the first three steps of the pathway. The
243-kDa CAD polypeptide is organized into separate structural domains (9, 10, 11) which have carbamyl phosphate
synthetase activity, aspartate transcarbamylase (EC 2.1.3.2), and
dihydroorotase activities (EC 3.5.2.3). Controlled proteolysis
studies(9, 10, 11, 12, 13, 14) and
determination of the amino acid sequence of the hamster CAD polypeptide (15, 16, 17, 18) showed that
carbamyl phosphate synthesis involves two distinct domains, a 120-kDa
CPSase synthetase domain and a 40-kDa glutaminase (GLNase) domain. Both
of the mammalian domains have a strong sequence similarity
(40-50%) to their counterparts in E. coli and other
species(17) .
The E. coli CPSase subunits have been
dissociated and shown to function autonomously(5) . However,
there is extensive evidence(5, 19, 20, 21, 22, 23, 24) for a
functional linkage between the glutamine binding site of the small
subunit and the active site of the large synthetase subunit which
modulates their activities. Glutamine hydrolysis does not proceed at a
significant rate in the absence of bicarbonate and ATP. This functional
linkage, which minimizes the hydrolysis of glutamine when the other
substrates needed for carbamyl phosphate synthesis are limiting, is
also present in CAD. ATP and bicarbonate increase the k of the glutaminase reaction 14-fold by
increasing the rate of hydrolysis of the thioester intermediate (25) formed during the catalytic cycle.
We have recently cloned the glutaminase domain of CAD and expressed the protein in E. coli(26) . The purified protein had barely detectable catalytic activity but formed a fully functional hybrid with the E. coli CPSase synthetase domain indicating that the functional linkage between the glutaminase and synthetase domains had been restored. This observation suggests that the subunit interfaces must be nearly identical in the eukaryotic and prokaryotic proteins and that the interdomain linkage has been highly conserved throughout the course of evolution.
The glutaminase domain of all carbamyl phosphate synthetases is unusual in that it consists of a 21-kDa carboxyl half which is homologous to all of the trpG-type amidotransferases and a 19-kDa amino half which occurs only in this family of molecules(17, 27, 28, 29, 30, 31, 32, 33) . The carboxyl half or catalytic subdomain contains the active site cysteine that participates in glutamine hydrolysis. The function of the amino half of the CPSase glutaminase domain is unknown, although, in a recent study (34) , a series of point mutations and deletions were introduced into the E. coli CPSase small subunit. Removal of the amino-terminal third (residues 1-113) of the glutaminase subunit prevented the association with the CPSase synthetase subunit, suggesting that this region is required to stabilize the complex. Thus, the amino half of the glutaminase domain has been designated the interaction subdomain.
As reported here, we have cloned and expressed the two halves of the hamster CAD glutaminase domain to determine whether they are separately folded subdomains with distinct functions. This study showed that all of the residues required for glutamine binding and hydrolysis are present in the catalytic domain and that the interaction domain, in addition to its role in maintaining the physical association of the glutaminase and synthetase domain, also participates in the interdomain signaling.
Figure 1: Subdomain structure of the CAD glutaminase domain. The glutaminase domain consists of residues 1-366 on the amino end of the CAD polypeptide. The 40-kDa domain is composed of two putative subdomains, the interaction and the catalytic subdomains. The interaction domain extends from residue 1 to 174. The catalytic subdomain, residues 175 to 365, contains three highly conserved regions (I, II, and III) that are common to all amidotransferases. Region IV is conserved only in the CPSases, but not found in other amidotransferases(2, 3) . Controlled proteolysis (14) cleaves the catalytic subdomain at residue 287 (protease site 1) more rapidly than the linker (protease site 2) connecting the GLNase and CPSase domains, suggesting that the catalytic subdomain may itself be composed of two smaller subdomains.
The recombinant, pJHN-GLN17, encoding the amino half of the hamster CAD glutaminase domain (Fig. 2), was obtained by deletion of pHG-GLN52(26) , a plasmid which codes for the entire 40-kDa glutaminase domain. The parent plasmid was restricted with StyI and EcoRI, and the 5` extensions were made flush using the Klenow fragment. The 3.9-kb fragment was gel-purified and religated to yield pJHN-GLN17, which contained a 539-bp cDNA insert encoding residues 1 to 180 of the CAD polypeptide. The coding sequence, thus, extends 6 residues into the putative catalytic subdomain. In addition, 3 residues derived from the vector are added to the carboxyl end of the protein. Expression of the recombinant, like the parental plasmid, is under control of the pyrBI promoter.
Figure 2:
Cloning of the CAD GLNase interaction and
catalytic subdomains. To clone the interaction subdomain (on the left), pHG-GLN52(26) , a plasmid encoding the CAD
GLNase domain (dark-shaded area) under the control of the pyrBI promoter (reg), was restricted with StyI and EcoRI, and the 5` extensions were made flush
using the Klenow fragment of DNA polymerase. The resulting 3.9-kb
plasmid pJHN-GLN17 contained a 539-bp cDNA fragment encoding the entire
interaction subdomain and six amino acid residues of the amino end of
the catalytic subdomain. In addition, three amino acids
(-Leu-Asn-Ser-COOH) derived from the vector are encoded prior to the
stop codon. The recombinant, pJHN-GLN17, is under control of the pyrBI promoter. To clone the catalytic subdomain (on the right), a 565-bp XhoII-SmaI fragment (dark-shaded area) was excised from the recombinant plasmid
pCAD142 (35) which contains most of the CAD coding sequence.
The fragment was inserted into the BamHI and EcoRI
sites of the vector pJLA503 after restriction and removal of the 5`
extensions of EcoRI cleavage site with mung bean nuclease. The
resulting recombinant pHG-GLN15 is under the control of the tandem
phage promoters (P
, P
) and has a
temperature-sensitive repressor gene (CI
) which allows
expression by heat shock. The plasmid encodes residues 175 to 365 of
CAD and has five amino acids (NH
-Met-His-Arg-Asn-Arg-) and
six amino acids (-Arg-Arg-Pro-Arg-Cys-Pro-COOH) derived from the vector
appended to the amino and carboxyl ends of the protein,
respectively.
The
sequence encoding the carboxyl half of the GLNase domain was excised
from the partial cDNA clone pCAD142(35) , by restriction with XhoII and SmaI (Fig. 2). The insert was cloned
into the plasmid, pJLA503, a heat shock vector under control of dual
promoters. The vector was cut with EcoRI and then with BamHI after removal of the 5`-EcoRI extensions with
mung bean nuclease. The resulting recombinant, pHG-GLN15, has a 565-bp
insert corresponding to residues 175 to 365 of the CAD polypeptide.
When expressed, the recombinant protein has 5 and 6 amino acid residues
derived from the vector added to the amino and carboxyl ends,
respectively.
For expression of the interaction subdomain, pJHGLN-17 transformants were grown in minimal media containing limiting uracil (12 µg/ml). The pyrBI promoter is derepressed when the uracil is exhausted (16-18 h), and a new 19-kDa protein began to accumulate in the extracts. This species was not found in extracts of cells transformed with the vector, pEK81. Immunoblots (Fig. 3) probed with hamster CAD antibodies confirmed that this polypeptide was of mammalian origin. The interaction subdomain was purified by DEAE-Sephacryl chromatography. We typically obtained 1-2 mg of the nearly homogeneous protein from a 500-ml culture of transformed cells.
Figure 3:
Expression and purification of the CAD
GLNase interaction subdomain. The plasmid pJHN-GLN17, which encodes the
GLNase interaction subdomain, was transformed into the strain EK1104 (lane 1) and the Lon protease-deficient strain L673 (lane
2). The induction followed the protocol described under
``Experimental Procedures.'' Cell extracts were prepared by
suspending 6 ml of packed cells (50 mg of cell protein) in 0.1 ml of 5
SDS-sample buffer and 0.4 ml of 50 mM Tris HCl, pH
7.4, 5% glycerol, 1 mM DTT. The suspension was immediately
heated to 85 °C for 15 min, then sonicated briefly, and 50-µl
aliquots were loaded onto a 7.5-15% gradient SDS-gel. This figure
shows the SDS-polyacrylamide gel (panel A) and the
corresponding immunoblot (panel B) of the pJHN-GLN17/EK1104 (lane 1), pJHN-GLN17/L673 (lane 2) transformants, and
EK1104 and L673 cells transformed with the vector only (lanes 3 and 4, respectively). An SDS-polyacrylamide gel of 1
µg of partially purified interaction domain (panel C) is
also shown.
For the expression of the catalytic subdomain, pHG-GLN15-transformed cells were induced by heat shock (see ``Experimental Procedures''). SDS-gel electrophoresis showed that extracts of pHG-GLN15 transformants contained a new species with a molecular mass of 21 kDa, that was not present in extracts of cells transformed with the vector. The recombinant protein reacted specifically with CAD antibodies indicating that it was part of the CAD polypeptide. Moreover, extracts of cells transformed with pHG-GLN15, but not the untransformed cells, had high glutaminase activity (Table 1). The protein was purified 667-fold (Table 1) by chromatography (Fig. 4) on a DEAE-Sephacryl column followed by Sephadex G-200 gel filtration. A 500-ml culture usually yielded 30 µg of nearly homogeneous catalytic subdomain.
Figure 4: Expression and purification of the CAD GLNase catalytic subdomain. The recombinant, pHG-GLN15, which encodes the GLNase catalytic subdomain, was transformed into the Lon protease-deficient E. coli strain, L673(37) , and induced as described under ``Experimental Procedures.'' Cell extracts were prepared as described in the legend to Fig. 3, from 6 ml of packed cells (60 mg of cell protein). The expression of the 21-kDa catalytic subdomain at 35, 90, and 150 min after heat shock (lanes 1-3) was shown by SDS-gel electrophoresis (panel B) and immunoblotting (panel D) of the pHG-GLN15 transformants. This species was not seen in extracts of cells transformed with the vector (lanes 4-6). For comparison, the SDS-polyacrylamide gel (panel A) and immunoblot (panel C) of EK1104 cells transformed with pHG-GLN52, which encodes the 40-kDa full-length GLNase domain, are also shown as is an SDS gel of 0.2 µg of the purified GLNase catalytic subdomain (panel E).
In
contrast, the purified catalytic subdomain was hyperactive (Fig. 5). Excision of the interaction domain increased the k to 5.73 s
, a value that is
347-fold higher than that of the isolated full-length GLNase domain,
and restored the K
to the normal value observed
for the intact CAD complex. As expected, the isolated GLNase
interaction domain had no detectable catalytic activity.
Figure 5:
Glutamine saturation curve of the GLNase
catalytic subdomain and of the GLNase activity of CAD. The activity
(-
) of the purified GLNase catalytic subdomain (0.2
µg) was assayed by the spectrophotometric method (see
``Experimental Procedures'') as a function of glutamine
concentration. For comparison, the glutamine saturation curve of 30
µg of CAD (
-
) is shown. In addition, a saturation
curve given by 30 µg of the isolated recombinant GLNase domain was
also obtained using the more sensitive HPLC method but is not shown
here. The kinetic parameters obtained for all three proteins are
summarized in Table 2.
Figure 6:
Gel filtration of the GLNase catalytic
subdomain and the E. coli CPSase synthetase subunit. The
purified E. coli synthetase large subunit (25 µg) was
incubated with the purified catalytic subdomain (5 µg) in 0.1 M KHPO
/KOH, pH 7.6, 1 mM EDTA, 1
mM DTT, 5% glycerol for 5 min at 4 °C and then
chromatographed on a 2.9
93 cm Sephacryl S-300 gel filtration
column. The column was pre-equilibrated and eluted with the same
buffer. NH
-dependent CPSase assays and SDS-gel
electrophoresis showed that the E. coli CPSase synthetase
subunit eluted in fractions 62 through 76. The SDS gel showed that the
catalytic subdomain eluted in fractions 78 through
80.
Figure 7:
Titration of the E. coli CPSase
synthetase subunit. Increasing amounts (0.05-6 µg) of the
purified mammalian glutaminase catalytic subdomain were incubated with
1.95 µg of the purified E. coli CPSase synthetase subunit
in 0.1 ml of 0.05 M Tris-HCl, pH 7.4, 1 mM DTT, 5%
glycerol for 5 min at room temperature. The samples were assayed for
glutamine-dependent carbamyl phosphate synthetase activity
(-
) as described under ``Experimental
Procedures.'' The ordinate represents fraction of maximum
observed activity. The molar ratio of the catalytic subdomain to the E. coli synthetase subunit was calculated assuming a molecular
mass of 20 and 120 kDa, respectively. The titration curve of the E.
coli synthetase subunit with the full-length GLNase domain,
carried out under identical conditions (
-
) taken from
Guy and Evans(26) , is also shown for
comparison.
This experiment indicated that the interactions
between the GLNase catalytic subdomain and the CPSase synthetase domain
are relatively weak, and that the complex, once formed, is not fully
functional. Glutamine saturation curves of this hybrid (Fig. 8)
gave the same K for glutamine, but the k
was 17-fold lower than the value obtained for
the hybrid formed from the complete GLNase domain.
Figure 8:
Kinetics of mammalian E. coli hybrids CPSase. The mammalian catalytic domain (2 µg) was
incubated at an equimolar ratio with the E. coli CPSase
synthetase subunit (10 µg), and the glutamine-dependent CPSase
activity was measured as a function of glutamine concentration (lower panel) at 5 mM [C]sodium bicarbonate (0.8
µCi/µmol) and 20 mM ATP. The saturation curve for the
hybrid of the full-length mammalian GLNase domain and the E. coli CPSase large subunit (upper panel), taken from Guy and
Evans(26) , is shown for comparison. The ordinate represents the specific activity of the complex expressed per mg
of the E. coli CPSase synthetase subunit. The V
of the hybrid of the mammalian GLNase
catalytic subdomain with the E. coli CPSase synthetase subunit
was 0.191 µmol/min/mg, while the corresponding hybrid formed with
the full-length mammalian GLNase domain is 3.21
µmol/min/mg.
Figure 9:
Sucrose gradient of the interaction domain
complex with the E. coli CPSase synthetase subunit. To obtain
the GLNase interaction subdomain, pJHN-GLN17/L673 transformants were
grown as described under ``Experimental Procedures.'' The
cell pellet (0.1 g) was resuspended in 0.5 ml of 0.2 M potassium phosphate, pH 7.6, 1 mM EDTA, and sonicated.
The extract was then centrifuged at 16,000 g for 20
min at 4 °C. The supernatant (0.150 ml, 3 mg of protein) was
incubated with the purified E. coli synthetase subunit (20
µg) in a total volume of 0.2 ml for 5 min at 25 °C. The
reaction mixture was then fractionated by sucrose gradient
centrifugation at 190,000
g for 9 h at 4 °C. The E. coli CPSase synthetase subunit (20 µg) was centrifuged
in a parallel gradient. The gradients containing the mixture of the
mammalian GLNase interaction subdomain and the E. coli CPSase
synthetase subunit (
-
) and the E. coli CPSase synthetase subunit alone (
-
), were then
fractionated and assayed for ammonia-dependent CPSase activity as
described under ``Experimental Procedures.'' An immunoblot of
the hybrid fractions using anti-CAD serum showed the presence of a
19-kDa fragment corresponding to the interaction subdomain
co-sedimenting with the E. coli synthetase
subunit.
When the cDNA sequences encoding the two halves of the CAD amidotransferase domain were cloned and expressed in E. coli, stable, soluble proteins were produced, which, when isolated, were found to have specific functions. This result showed that the glutaminase domain of the CAD CPSase, and, by inference, the other members of this family, consists of two independent subdomains which fold and function autonomously. The chain segment connecting the catalytic and interaction subdomains is probably buried within the molecule since no proteolytic cleavage site was found (14) at the junction between the two subdomains.
The complete CAD GLNase
domain has very low glutaminase activity as a consequence of a 46-fold
increase in the K and an 8.5-fold lower k
compared to the same domain present in the
intact CAD complex. Thus, when isolated from the remainder of the
molecule, the apparent second order rate constant k
/K
is reduced 392-fold.
Similarly, when the subunits of E. coli CPSase were
dissociated(5) , there was a 300-fold increase in the K
for glutamine. These authors suggested that
there may be additional residues on the large synthetase subunit which
are involved in glutamine binding. However, we show here that the
isolated catalytic subdomain has the same K
for
glutamine as the intact CAD complex and a k
which exceeds that of CAD activated by ATP and bicarbonate. The
differences are even larger when the activity of the catalytic domain
is compared to the isolated complete GLNase domain. The k
of the catalytic subdomain is 347-fold higher,
and the K
is 40-fold lower than the corresponding
values for the GLNase domain. Thus, excision of the interaction
subdomain from the CAD glutaminase results in a 14,000-fold increase in
the apparent second order rate constant for glutamine hydrolysis. This
result demonstrates that all of the residues necessary for glutamine
binding and catalysis are located within the catalytic subdomain. It is
more likely that the suppression of the glutaminase activity is a
consequence of the functional linkage between the GLNase and CPSase
domains that is necessary to avoid the hydrolysis of glutamine when the
other substrates required for the synthesis of carbamyl phosphate are
limiting.
The interaction domain binds to the E. coli synthetase domain forming a complex which is stable enough to detect by sucrose gradient centrifugation. This result is consistent with the results of Guillou et al.(34) who showed that deletion of 113 residues from the amino end of the E. coli GLNase subunit prevents its association with the synthetase subunit. Thus, the interaction domain is crucial for the formation of a stable complex between the glutaminase and synthetase domains or subunits. In contrast, we were unable to detect a stable complex of the catalytic subdomain and the E. coli CPSase synthetase subunit, indicating that the interaction between these domains are weak. Nevertheless, the titration curve (Fig. 7) and the steady state kinetics (Fig. 8) clearly show that interactions between the catalytic subdomain and the the CPSase synthetase domain do exist.
Analysis of the titration curve suggests that when the mammalian
GLNase catalytic subdomain is mixed in a one to one molar ratio with
the E. coli CPSase synthetase subunit, only 5% of the E.
coli subunits have bound the GLNase domain. Glutamine-dependent
CPSase assays measure only the activity of the complex since the free
unbound CPSase synthetase has no glutamine-dependent activity. Taking
this factor into account, we can calculate the steady state kinetic
parameters (Table 3) of the complex between the catalytic
subdomain and the synthetase subunit. The K for
glutamine is similar to that of the hybrid formed with the complete
mammalian GLNase domain. However, the k
for the
glutamine-dependent CPSase activity is 8.5-fold lower indicating that
while the catalytic subdomain binds, albeit tenuously, to the CPSase
domain and can transfer ammonia to the synthetase subunit, the
functional linkage is not restored.
This study, therefore, shows that the interaction domain has two functions, 1) it forms strong noncovalent interactions with the CPSase synthetase domain that are necessary for the functional interactions, and 2) it interacts with the GLNase catalytic subdomain to decrease glutamine binding and suppress the high activity of the subdomain.
The low affinity for glutamine
and a low turnover number of the isolated GLNase domain can thus be
attributed to inhibitory effects exerted by the interaction domain.
When the glutaminase domain associates with the synthetase domain, this
inhibition is partially relieved. The K is
restored to normal levels, and the k
increases
from 0.0165 to 0.140. However, only in the presence of ATP and
bicarbonate does the turnover number approach the value observed for
the isolated catalytic subunit.
We propose that the interaction domain is an important element of the functional linkage between the CPSase glutaminase and synthetase domains or subunits. In this model, illustrated schematically in Fig. 10, the GLNase domain can exist in two states. In the absence of the other CPSase substrates, ATP and bicarbonate, the domain is in the ``off'' state. The activity is low because the interaction domain reduces the rate of the breakdown of the thioester intermediate which accumulates and therefore prevents the enzyme from embarking on another round of catalysis.
Figure 10: Schematic representation of carbamyl phosphate synthetase. The diagram is a schematic representation of the proposed model for the linkage between the GLNase and CPSase domains. In the absence of ATP and bicarbonate, the glutaminase domain has low activity (off state). The CPSase domain interacts with both GLNase subdomains although the interactions with the catalytic subdomain are weak and not sufficient to form a stable complex. The thioester forms and ammonia is released, but its breakdown is suppressed by the interaction subdomain. The thioester accumulates and glutamine hydrolysis is very slow. In the presence of ATP and bicarbonate (on state), a conformational change is induced within the CPSase domain which is then transmitted to the GLNase domain. The interaction domain no longer impedes the hydrolysis of the thioester. The active enzyme is regenerated for another round of catalysis, and ammonia is provided for carbamyl phosphate synthesis.
The binding of ATP and bicarbonate to the synthetase domain induces a conformational change which is transmitted to the interaction domain, with which it intimately interacts, and triggers the ``on'' state. These structural changes disrupt the attenuating interactions between the catalytic and interaction subdomains and relieves the inhibition. The thioester once formed rapidly breaks down and the enzyme turns over.
The mechanism by which the interaction domain
suppresses the activity of the catalytic subdomain remains unknown.
However, as we suggested previously(14) , the catalytic
subdomain may in turn be comprised of two smaller structural units,
since controlled proteolysis experiments demonstrated a hypersensitive
cleavage site characteristic of an interdomain linker or hinge located
in the middle (residue 287) of the subdomain. The two halves carry a
very different charge so they may associate by electrostatic
interactions. The calculated pI of the 12.8-kDa segment (residues
174-287) on the amino end is 9.7, while the 8.5-kDa segment
(residues 288-365) on the carboxyl side has a pI of 4.1. It is
interesting that the active site cysteine, Cys-252 is present in the
amino half while the other 2 active site residues identified thus far
in CAD, ()His-336 and Glu-338 are located in the carboxyl
half of the catalytic subdomain. Moreover, there is a 10-residue,
highly conserved sequence, region IV (residues 291-300), located
in the carboxyl half of the catalytic domain immediately adjacent to
the cleavage site. Since this sequence is conserved only in the
CPSases, not in any other amidotransferases, we had suggested that
region IV interacts specifically with the CPS synthetase domain and
mediates interdomain signaling. In E. coli CPSase(49) , mutation of a region IV histidine residue
(corresponding to His-295 in the CAD sequence) reduces glutamine
binding. Moreover, saturating glutamine protects the catalytic
subdomain from proteolysis, (
)which supports the idea that
this putative hinge is located near the glutamine binding site or
undergoes a change in conformation when glutamine binds. An attractive
idea, which remains to be tested, is that the interaction domain may
alter the juxtaposition of the two halves of the catalytic subdomain
and thus prevents the active site from fully forming or assuming the
optimal conformation for catalysis.