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INTRODUCTION |
Creatine and its phosphorylated form provide a dynamic reservoir
of high energy phosphate in several tissues, most prominently in
skeletal muscle and heart (1). Most vertebrates are able to synthesize
creatine de novo from L-arginine and glycine (2) by the successive action of L-arginine:glycine
amidinotransferase (AT)1
(transamidinase, EC 2.1.4.1) and
S-adenosylmethionine:guanidinoacetate N-methyltransferase (guanidinoacetate methyltransferase, EC
2.1.1.2) (1), whereas most invertebrates must obtain creatine from
their diet. In higher vertebrates, creatine is synthesized primarily in
the liver, pancreas, and kidney, transported by the bloodstream and
taken up by Na+-dependent creatine transporters
into muscle and neural tissues (3, 4), where the majority of the
creatine/phosphocreatine pool is located (1).
The de novo biosynthesis of creatine is controlled at the
level of AT (1), which catalyzes the rate-limiting step of creatine biosynthesis: the transfer of an amidino group from
L-arginine to glycine, resulting in L-ornithine
and guanidinoacetic acid, the immediate precursor of creatine. AT is
regulated in a variety of ways, including product inhibition of its
catalytic activity by ornithine (5), end product repression of its
synthesis at a pretranslational level by creatine (6-10), induction by
growth hormone and thyroxine (9, 11), and repression during embryogenic development (12-14). Deficiencies of L-ornithine:2-oxoacid
aminotransferase (EC 2.6.1.13) result in hyperornithinemia (15), which
is associated with type II muscle fiber atrophy and gyrate atrophy of
the choroid and retina, a disease that progressively leads to blindness
(16). Because ornithine is a strong competitive inhibitor of AT, its 10-20-fold increased plasma concentration in hyperornithinemia results
in inhibition of AT, suggesting impaired de novo creatine biosynthesis as the cause of the aforementioned pathogenesis of the eye
and muscle (16, 17).
AT exhibits a rather broad substrate specificity and can utilize a wide
variety of amidino donors, such as L-canavanine,
4-guanidinobutyrate, 3-guanidinopropionate, and hydroxyguanidine, and
amidino acceptors, such as L-canaline, 4-aminobutyrate,
3-aminopropionate, and hydroxylamine, in addition to the physiological
substrates (1). Biochemical data suggested a ping-pong mechanism for
the transamidination reaction (18, 19), including a transient covalent
attachment of an amidino group to Cys407 (20-22). The
crystal structure of human AT at high resolution revealed that the
nucleophilic Cys407 is located at the bottom of an narrow
active site channel (23). The enzyme adopts open and closed
conformations during the reaction cycle. The crystal structures of AT
in complex with L-arginine and L-ornithine
showed that binding is accompanied by large conformational changes,
with shifts of up to 5 Å for the 300-flap and for helix H9 compared
with the unliganded enzyme (Fig. 1) (23). However, highly homologous
amidinotransferases are also found in certain strains of
Streptomyces sp., in which they catalyze two
transamidination reactions in the biosynthesis of the streptomycin
family of antibiotics (24-27). These amidinotransferases lack the
flexible helix H9 in human AT as the most prominent difference,
indicating distinctly different ligand and intermediate binding. The
crystal structure of L-arginine:inosamine-phosphate
amidinotransferase StrB1 from Streptomyces griseus (StrB1)
was solved recently at 3.1 Å (28) and revealed a fold closely related
to AT. However, major changes were found in loops surrounding the
active site, resulting in an open and solvent exposed cavity. These
differences can be largely attributed to the deletion of AT helix H9
and to the deletion of one residue in the AT 300-loop. The conformation
of the 300-loop implies that StrB1, in contrast to AT, adopts a single
conformation during catalysis.
The aim of the work described here was to establish a role for the
induced-fit movements of AT and in particular for AT helix H9. In
addition, the properties of a ligand molecule inducing the large
conformational changes upon binding should be clarified. The crystal
structures of AT in complex with the amidino acceptor analogs
-aminobutyric acid and
-aminovaleric acid on the one hand, and
the amidino donor analogs L-alanine,
L-
-aminobutyric acid, and L-norvaline on the
other hand (Fig. 2), revealed two distinct binding modes. A third
binding mode was found for glycine, reflecting the rather broad
substrate specificity of AT. The ability of these compounds to trigger
the induced-fit movements was investigated and led to the
identification of the required chemical groups. A multiple sequence
alignment revealed that residue Met302 and helix H9 are
deleted in the bacterial amidinotransferases. To investigate the role
of helix H9 and of residue Met302, we prepared appropriate
deletion mutants of human AT and examined their kinetic, thermodynamic,
and structural properties. The implications of these results for the
structure and the induced-fit mechanism of AT are discussed.
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EXPERIMENTAL PROCEDURES |
Protein Expression and Purification--
Wild-type AT, the AT
deletion mutants AT
M302 and AT
11, and StrB1 were overexpressed as
N-terminal hexahistidine fusion proteins and purified by
nitrilotriacetate-agarose affinity chromatography as described
previously (20, 28). In order to obtain highly pure StrB1 for
spectroscopic experiments, an additional purification step was
essential. Briefly, the pooled fractions from the
nitrilotriacetate-agarose affinity column were dialyzed against Buffer
A (30 mM Tris-HCl, pH 7.4, 1 M ammonium
sulfate, 0.5 mM EDTA, 0.5 mM GSH) and applied at a flow rate of 2 ml/min to a 24-ml column of phenyl-Sepharose 6 FF
(Amersham Pharmacia Biotech) equilibrated with Buffer A. After washing
with Buffer A, the enzyme was eluted with a linear salt gradient
(0.8-0 M) of ammonium sulfate in Buffer B (30 mM Tris-HCl, pH 7.4, 0.5 mM EDTA, 0.5 mM GSH). The fractions containing StrB1 were combined and
dialyzed against 30 mM HEPES-NaOH, pH 7.7, 0.5 mM EDTA, and 0.5 mM GSH. Purity of the enzyme
preparations were assessed by SDS-PAGE through 12% polyacrylamide gels
(29) followed by staining with Coomassie Briliant Blue R-250. The
identity of the isolated proteins was verified by N-terminal
sequencing, mass spectrometry, and enzymatic activity. The deconvoluted
mass spectra revealed major peaks for wild-type AT of 45,812 Da
(electrospray ionization MS, calculated mass, 45,777 Da), for AT
M302
of 45,659 Da (electrospray ionization MS, calculated mass, 45,646 Da),
for AT
11 of 44,540 Da (matrix assisted laser desorption/ionization MS, calculated mass, 44,491 Da), and for StrB1 of 40,170 Da
(electrospray ionization MS, calculated mass, 40,163 Da). The yields of
electrophoretically homogeneous protein per liter of culture were 2-3
mg for AT and AT
M302 and 0.2-1 mg for AT
11 and StrB1.
Mutagenesis--
For preparation of the mutant AT
M302 (lacks
Met302) the QuikChangeTM mutagenesis kit
(Stratagene, Heidelberg, Germany), the plasmid pRSETAT38H (20), and the
primers 5'-GGTAGCATCAATATGGGGATTGGGATC-3' and
5'-GATCCCAATCCCCATATTGATGCTACC-3' were used. The deletion mutant
AT
11 (lacks amino acids Ile229-Ala239) was
prepared using the ExSiteTM mutagenesis kit (Stratagene)
with primers 5'P-GGGATAATCCTGGTTATAAAGC-3' and
5'P-GCTCAGGGAAAATTTGTGACAACTGAATTCGAGCCATGC-3' (both
primers contained a 5'-phosphate group). The primers were purchased
from MWG-Biotech (Ebersberg, Germany). The deletions were verified by
DNA sequencing of the whole coding region in both directions.
Protein Determination and Activity Measurements--
The protein
concentration was determined spectrophotometrically using a specific
absorption coefficient at 280 nm of 2.0 cm2·mg
1 for AT and its variants, and 1.46 cm2·mg
1 for StrB1. The activity of AT and
StrB1 was measured by the method of Van Pilsum et al. (30),
by incubating the enzyme with 20 mM L-arginine
and 20 mM of glycine or hydroxylamine, as amidino donor and
acceptor, respectively, and determining the formed
L-ornithine by a ninhydrin color reaction. One enzyme unit
is defined as the amount of enzyme that releases 1 µmol of
L-ornithine/h at 37 °C.
Crystallization--
The purified proteins were concentrated by
ultrafiltration (Centricon-30, Amicon) to approximately 13 mg/ml.
Crystals of AT and AT
M302 were grown by vapor diffusion using the
hanging drop method. The droplets were made from 7 µl of a protein
solution (13 mg/ml in 30 mM HEPES-NaOH, 0.5 mM
EDTA, 0.5 mM GSH) and 14 µl of a reservoir solution (3%
(w/v) polyethylene glycol 6000, 40 mM HEPES-NaOH, pH 7.0)
and equilibrated against the reservoir solution.
Tetragonal-bipyramidal-shaped crystals of AT (approximately 0.7 × 0.4 × 0.4 mm) appeared after 2 days. Crystals from the mutant AT
M302 grew from precipitated protein within 5 days. AT
M302 crystallized isomorphously to the wild-type enzyme in the tetragonal space group P43212, with lattice constants of
a = b = 83.71 Å, c = 200.41 Å, and a = b = c = 90° (Table I). The
asymmetric unit contained one molecule with a solvent content of 68%
(Vm = 3.83 Å3/Da) (31).
Soaking Experiments--
Crystals were transferred from the
mother liquor to droplets of harvesting buffer (20% polyethylene
glycol 6000, 267 mM HEPES-NaOH, pH 7.0) containing either
glycine (0.5 M),
-aminobutyric acid (0.1 M),
-aminovaleric acid (1.0 M), L-alanine (0.5 M), L-
-aminobutyric acid (0.5 M), or L-norvaline (saturated), and soaked for
15-50 h. The chemical structures of the soaked compounds are presented in Fig. 2.
Data Collection and Processing--
X-ray measurements were
performed on a MAR Research imaging plate detector system at 18 °C
mounted on a Rigaku RU200 rotating anode x-ray generator with
=
CuK
= 1.542 Å. Images were processed with MOSFLM (32)
and data were scaled and reduced using ROTAVATA/AGROVATA/TRUNCATE (33).
The statistics of data collection are given in Table I.
Refinement and Quality of the Final Model--
As the crystals
of the AT deletion mutant AT
M302 and of all complexes were
isomorphous with those of the wild-type protein, the wild-type
structure was used as a model for initial Fourier (2Fo
Fc) and difference
Fourier (Fo
Fc) maps.
Subsequently, the substrates and inhibitors were fitted to the electron
density using FRODO (34) on an ESV-30 Graphic system workstation (Evans
& Sutherland, Salt Lake City, UT), and the model was refined with XPLOR
(35) using the parameter set of Engh and Huber (36). The final model
comprises residues 64-423. The region from residue 230 to residue 242 displays average main chain B values, which are about 10 Å2 (AT in closed conformation) and 45 Å2 (AT
in open conformation) above the B value for the whole protein and
correlate with poor electron density. Water molecules were built into 1
peaks of the (2Fo
Fc) map. Water molecules with B factors above 50 Å2 were removed
from the model prior to the last refinement. The models show good
geometry with R-factors below 20% and root mean square deviations for
a bond length of 0.010-0.011 Å and for bond angles of 1.56-1.62°.
Two of the three Ramachandran outliers (37) (Met184 and
His303) are well defined and are also observed in the
wild-type AT structure (23), whereas Ser231 is poorly
defined and is observed as outlier solely in the structures adopting an
"open" state. The final refinement statistics and stereochemical
parameters are presented in Table I.
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Table II
Sedimentation analysis of AT M302, AT 11, and StrB1
Given the dimeric quaternary structure, the calculated molecular masses
for AT, AT M302, AT 11, and StrB1 are 91.8, 91.6, 89.2, and 80.3 kDa, respectively. ND, not determined.
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Circular Dichroism Measurements--
Far-UV circular dichroism
spectra were recorded on a Mark-IV (Jobin Yvon) spectropolarimeter. The
spectra were recorded from 195 to 250 nm in a thermostated 0.01-cm
quartz cuvette at 10 °C. A protein concentration of about 0.15 mg/ml
in phosphate-buffered saline (39), pH 7.4, 0.5 mM EDTA, and
1 mM GSH was used. All spectra were accumulated four times
and corrected for the buffers. Mean molar residue ellipticities
(
R) were calculated based on a mean residue molecular
weight of 113. The thermal unfolding curves were determined by
monitoring the changes in dichroic intensity at 216 nm as a function of
temperature. The measurements were carried out in the temperature range
of 10-80 °C with a temperature gradient of 0.5 °C/min using an
oil-thermostatted cylindrical 0.01-cm quartz cell connected to a
thermoprogrammer. The Tm values were derived by
fitting the temperature-induced unfolding curves using the
Gibbs-Helmholtz equation,
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(Eq. 1)
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where
G is the free energy of unfolding,
Tm is the temperature at which 50% of the protein
molecules are unfolded,
Hm is the enthalpy of
unfolding at Tm, and
Cp is the heat
capacity. A
Cp value of 2.7 kcal·mol
1K
1 was estimated from the amino
acid composition (38). Because the thermal transitions of all studied
proteins were irreversible, a quantitative evaluation of thermodynamic
parameters was not possible.
Size Exclusion Chromatography--
Size exclusion chromatography
was performed using a Superose 6 PC 3.2/30 column mounted on a
SMARTTM system (Amersham Pharmacia Biotech). For all
measurements a flow rate of 40 µl/min and phosphate-buffered saline,
pH 7.4, 0.1 mM EDTA, 0.5 mM dithiothreitol as
buffer was used. The following marker proteins were used for
calibration: bovine serum albumin (66 kDa), ovalbumine (45 kDa),
carbonic anhydrase (29 kDa), and ribonuclease A (13.7 kDa).
Analytical Ultracentrifugation--
Sedimentation velocity and
sedimentation equilibrium experiments were performed in a Beckman
Spinco Model E analytical ultracentrifuge equipped with a photoelectric
scanner. Double-sector cells with sapphire windows were used in an An-G
rotor. Initial protein concentrations were about 0.2 mg/ml (AT
M302
and AT
11) and 0.27 mg/ml (StrB1), corresponding to an initial
absorbance at 280 nm of 0.4. Prior to the experiments, the protein
samples were equilibrated against phosphate-buffered saline, pH 7.4, 0.5 mM EDTA, 0.5 mM dithiothreitol. Sedimentation coefficients were determined at 20 °C and 44,000 rpm,
plotting lnr versus time. The scanning wavelength
was 280 nm. The partial specific volume was calculated using the
partial specific volumes of the component amino acids, yielding a value of 0.735 ml/g. The solvent density was estimated to be 1.000 g/ml. High-speed sedimentation equilibria measurements (40) were performed at
22 °C and 16,000 rpm. The molecular weight was calculated from lnc versus r2 plots using
a computer program written by G. Böhm (University of Regensburg).
The scanning wavelengths were 230 and 280 nm.
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RESULTS AND DISCUSSION |
Crystal Structures of AT in Open Conformation--
The crystal
structures of AT and of its complexes with L-arginine and
L-ornithine have been described (23), and they show that AT
exists in at least two different conformations: (i) a closed
conformation of ligand-free AT, and (ii) an open conformation induced
by the binding of L-arginine (to AT mutant C407S) and L-ornithine (Fig. 1). In
order to evaluate the molecular basis of these ligand-induced
conformational changes, the structures of the binary complexes of AT
with the amidino donor analogs L-norvaline, L-
-aminobutyric acid, and L-alanine (Fig.
2) were solved at resolutions of 2.4, 2.5, and 2.3 Å, respectively. These amino acids differ in the number
of side chain carbon atoms, and they are not substrates because they
lack the terminal amino group. In the presence of substrate
concentrations of 10 mM, only L-norvaline was
found to inhibit amidine transfer (41, 42). The x-ray structures of
L-norvaline, L-
-aminobutyric acid, and
L-alanine provide a clue for the inhibition mechanism at
atomic resolution. All these ligands induce the transition from the
closed to the open state and bind similar to the
L-ornithine and L-arginine co-crystal structures (Fig. 1). The carboxylate and
-amino groups of these compounds would clash with the side chain of Asn300 in the
closed conformer. This leads to a displacement of the 300-flap
(residues
Asp298-Pro299-Asn300-Pro301)
toward helix H9 (residues Val232-Gln241). A
concomitant repositioning of helix H9 prevents steric collision of
Pro299 with His236 and of Asn300
with Arg235. The shifts are as large as 5.1 Å for the
C
position of Pro299 and about 3.3 Å for
the C
positions of helix H9 (Fig. 1). These motions open
the active site and may facilitate ligand binding and dissociation of
reaction products. In agreement with the canonical binding, the
carboxylate oxygen OT1 of L-norvaline accepts a
hydrogen bond from Ser355 O
(2.8 Å) and is
fixed via a bifurcated hydrogen bond to N
1 and
N
2 (both 2.9 Å) of Arg322 (Fig.
3A). The amide nitrogen of
Ser355 donates a hydrogen bond to the OT2
carboxylate oxygen (2.8 Å). The
-amino group is a hydrogen bond donor to the carbonyl oxygen of Met302 (2.8 Å). In
contrast to the substrate binding residues, the 300-flap was not
unambigously defined in the L-alanine complex, indicating a
partial occupation of two (open and closed) positions. This could be
attributed to a decreased binding affinity of L-alanine due
to its small side chain. In accordance, L-norvaline is the best of these amino acid inhibitors and is identical to
L-ornithine in the length of the carbon side chain. In
contrast to L-alanine, L-ornithine is a strong
competitive inhibitor (Ki, 0.253 mM),
and the plasma L-ornithine levels in hyperornithinemia may be sufficient to inhibit AT, resulting in gyrate atrophy of the choroid
and retina (17).

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Fig. 1.
The ligand-induced structural changes of
AT. A, superimposition of helix H9 and of the 300-loop
in open conformation (blue) and "closed" AT
(yellow). Binding of an L-ornithine molecule
(green) would sterically interfere with the side chain of
Asn300 in the closed conformation and induces the
displacement of the 300-loop in a concerted fashion with H9.
B, comparison of the 300-loop and helix H9 of AT in the open
(blue) and closed (yellow) conformations. The
orientation is the same as in A. The open conformation is
stabilized by hydrogen bonds from Pro299 O and
Asn300 O 1 to the guanidino group of
Arg235 (dashed lines). The coordinates were
taken from Ref. 23, and the figure was produced with SETOR (46).
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Fig. 2.
Chemical formulas of compounds mentioned in
the text. The physiological substrates and products of
L-arginine:glycine amidinotransferase are shown. The
various analogs mentioned in the text are arranged in a group of
amidino donor analogs (which contain an -amino group) and a group of
amidino acceptor analogs (which do not contain an -amino
group).
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Fig. 3.
The binding modes of L-norvaline
(A), -aminovaleric acid (B), and glycine
(C) and the active site topology of the mutant AT M302
(D). A, the binding mode of
L-norvaline. AT is found in the open conformation. The
residue Asn300 is displaced upon binding of
L-norvaline (see Fig. 1B). B, the
-aminovaleric acid binding site. AT is found in the closed
conformation. Asn300 hydrogen bonds to the ligated
-aminovaleric acid molecule. C, the glycine binding site.
Only the hydrogen bonds to Ser354, Wat111, and
His303 are within hydrogen bond distances; the other
interactions indicated are in the range of 3.9 Å. For A-C,
the Fo - Fc difference electron
density maps contoured at the 1 level are shown in red
and superimposed on the refined models. D, active site
topology of mutant AT M302. The final 2Fo - Fc electron density map contoured at 1 (green) is superimposed on the refined model. For
comparison, the corresponding residues of closed AT are represented as
black sticks. A modeled L-norvaline inhibitor molecule is
shown in cyan. The deletion of residue Met302
results in a new side chain conformation of Asn300, which
allows binding without obvious steric restrictions. The figure was
produced with SETOR (46).
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Crystal Structures of AT in Closed Conformation--
The crystal
structures of unligated AT, as well as of AT in complex with the
amidino acceptor or analogs glycine,
-aminobutyric acid, and
-aminovaleric acid (Fig. 2), were found in closed conformation.
-Aminobutyric acid and
-aminovaleric acid bind differently to L-norvaline, L-
-aminobutyric acid, and
L-alanine. The carboxylate group of the ligand is shifted
1.2 Å away from Asn300, which allows binding without
inducing conformational changes (Fig. 3B). The carboxylate
oxygen OT1 of
-aminovaleric acid is a hydrogen bond
acceptor to N
1 of Arg322 (3.2 Å).
OT2 shares a hydrogen bond to Ser355
O
(2.5 Å), to the amide nitrogen of Ser355
(2.9 Å), and to N
2 of Asn300 (2.9 Å). A
buried water molecule bridges OT1 (2.8 Å) to the side
chain of Thr307 (2.8 Å), and to Ala306 N (2.9 Å) and His303 O (2.7 Å), respectively. The
-amino
group of
-aminovaleric acid is surrounded by Asp305,
Cys407, His303, and Asp170. A
comparison of the structures in complex with
-aminobutyric acid and
-aminovaleric acid with L-norvaline,
L-
-aminobutyric acid, and L-alanine revealed
that the
-amino group and not the number of carbon atoms is the
principal determinant for the movement of the 300-flap in concert with
helix H9.
For glycine, a completely different binding mode was observed. Its
carboxylate group is not involved in ion pair formation with
Arg322 but makes a bifurcated hydrogen bond to
His303 N
1 (3.4 Å) and to a water molecule
(3.0 Å), which hydrogen bonds to Tyr164 OH (3.1 Å) (Fig.
3C). The amino group donates a hydrogen bond to
Ser354 O
(3.3 Å) and is 4.6 Å apart from
the nucleophilic cysteine 407. A simultaneous binding of
L-arginine and glycine is not possible, in accordance with
a double-displacement (ping-pong) mechanism.
Structure of AT
M302--
A comparison of the amino acid
sequences of AT and StrB1 revealed a one residue deletion at position
302. The corresponding deletion
M302 was introduced into the AT gene
by site-directed mutagenesis, and the variant was recombinantly
expressed and crystallized. This variant differs from the wild-type
structure only at the site of the Met302 deletion, which
results in a new path for residues
(Asn300-Pro301-Met302), whereas the
flanking residues Pro299 and His303 are at
topologically identical positions to the wild-type structure. Most
remarkably, the side chain of Asn300 takes an alternative
position, which would not interfere with L-ornithine
binding, and in fact could contribute a hydrogen bond to the
-amino
group (Fig. 3D). The carbonyl oxygen of Pro301
of AT
M302 could substitutes for the carbonyl oxygen of the deleted residue Met302, accepting a hydrogen bond from the
-amino group. These findings suggest that AT
M302 does not switch
between an open and closed state but is permanently locked in the
closed conformation.
Evidence for Structural Integrity of AT
M302 and
AT
11--
The structural integrity of the mutants AT
M302 and
AT
11 was demonstrated by x-ray crystallography (for AT
M302),
analytical ultracentrifugation experiments, and circular dichroism
spectroscopy. The far-UV circular dichroism spectra of these mutants
are very similar. Sedimentation coefficients and molecular masses of
AT
M302 and AT
11, determined by analytical ultracentrifugation,
were found to be in good agreement with results for human kidney
L-arginine:glycine amidinotransferase (Table II) (43). The
sedimentation analysis shows that AT and its mutants AT
M302 and
AT
11, as well as StrB1, form homodimers and that the mutations did
not affect dimerization. However, gel filtration experiments for
wild-type AT, AT
M302, AT
11, and StrB1 yielded apparent molecular
masses between 52 and 56 kDa for all proteins, probably as a result of
adsorption effects on the size-exclusion column.
Kinetic Properties of AT
M302 and AT
11--
A kinetic
analysis of the deletion mutant AT
302 revealed an activity of about
0.15 units/mg, corresponding to a 100-fold decrease as compared with
the wild-type enzyme (specific activity of about 15 units/mg). Both AT
and AT
M302 displayed a background arginase activity of about 0.1 units/mg as reported previously (44), resulting from the hydrolysis of
the covalent enzyme amidine intermediate. As mentioned above, the new
side chain conformation of the key residue Asn300 may allow
binding of substrates without inducing conformational changes. It is
suggested that the main reason for the low enzymatic activity of this
mutant is the lack of ligand-induced conformational changes, which
somehow reduces the binding forces for products to accelerate amidine
transfer. This view is in agreement with the comparable low enzymatic
activity of 0.6 units/mg of StrB1, which naturally lacks helix H9 and
possesses a one-residue deletion in the 300-flap.
In order to investigate the role of helix H9, a mutant lacking 11 residues from Ile229 to Ala239 was prepared by
site-directed mutagenesis. Remarkably, the resultant deletion mutant
AT
11 showed a residual activity of 1.0 units/mg. This corresponds to
a 15-fold reduced activity and lies in the range of StrB1, which
naturally lacks helix H9. Obviously, the conformational flexibility of
helix H9 is important for efficient catalysis and may stabilize the
open conformation of the 300-flap. Indeed, the residue
Arg235 of H9 donates two hydrogen bonds to
Pro299 O and one to Asn300 O
1,
thereby fastening H9 to the 300-flap (Fig. 1B).
Additional evidence that the ligand-induced conformational changes
increase the catalytic activity stems from activity measurements of AT,
AT
M302, and AT
11 in the presence of L-arginine and
-aminovaleric acid as amidino donor and acceptor, respectively. The
activity of the wild-type enzyme was found to be reduced about 5-fold, reflecting weaker binding of
-aminovaleric acid and/or impaired dissociation. In contrast, no reduction of enzymatic activity was
observed for AT
M302 and AT
11. A likely explanation is that the
dissociation of L-ornithine from these closed variants is hampered and limits the overall velocity.
Stability Measurements--
In order to evaluate a possible role
of helix H9 in protein stability, temperature-induced transition curves
were measured for AT
11 and compared with AT and AT
M302. The
thermal transitions were irreversible and did not allow determination
of thermodynamic data. AT and AT
M302 show similar midpoints of
transition at Tm = 51.9 and 51.2 °C,
respectively. For AT
11, the Tm value (53.1 °C), as well as the cooperativity of the transition, is slightly increased (Fig. 4). These
results suggest that neither residue Met302 nor helix H9
adds to the stability in vertebrate AT.

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Fig. 4.
Thermal unfolding curves for AT ( ),
AT M302 ( ), and AT 11 (×). The change in dichroic
intensity at 216 nm as a function of temperature was monitored. The
heating rate was 0.5 °C/min. Measurements were done using 0.15 mg/ml
of protein in phosphate-buffered saline, pH 7.4, 0.5 mM
EDTA, 0.5 mM dithiothreitol. See under "Experimental
Procedures" for details.
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Implications for the Induced-fit Mechanism--
The crystal
structures of AT in complex with L-alanine,
L-
-aminobutyric acid, L-norvaline, glycine,
-aminobutyric acid, and
-aminovaleric acid showed three different
binding modes that lead to an understanding of the broad substrate
specificity. It is known from L-arginine analogs that only
L-arginine (chain number of carbon atoms (n) = 5) and L-canavanine (the guanidinoxy analog of arginine)
can serve as amidino donor. Longer and shorter arginine analogs, such
as homoarginine (n = 6),
-amino-
-guanidinobutyric acid (n = 4), and
-amino-
-guanidinopropionic acid
(n = 3) are not substrates for AT (1). These amino acid
derivatives are supposed to bind to AT via their
-amino and
carboxylate groups, but only compounds with the appropriate chain
length reach the nucleophilic cysteine and are able to transfer their
amidino groups. The same holds true for L-norvaline and
related inhibitors, such as L-
-aminobutyric acid, and
L-alanine, which bind in a similar manner but lack the
terminal guanidino/amino group. In contrast, the chain length is not
important for glycine analogs functioning as amidino acceptors as long
as they are shorter than n = 6. Glycine (n = 2),
-aminopropionic acid (n = 3),
-aminobutyric acid (n = 4), and
-aminovaleric
acid are substrates for AT, albeit inferior to glycine (1). We showed
that the amidino acceptor analogs
-aminobutyric acid
(n = 4) and
-aminovaleric acid (n = 5) bind differently to amino acid derivatives and do not induce the
displacement of the 300-loop and of helix H9. Acceptors with fewer than
n = 4 carbon atoms are proposed to use different
binding modes. This was observed for glycine, which binds in a
completely distinct manner, closer to the nucleophilic cysteine 407. It
was noted before (45) that arginine reacts with both AT and StrB1,
whereas glycine is not a substrate for StrB1. A comparison of the two active sites revealed that residues interacting with
L-arginine are all conserved but that most residues
involved in glycine binding are not conserved. The residues
Tyr164, Ser354, and Asn300 are
topologically replaced by residues His102,
Ala224, and Cys279 in StrB1, disrupting the
hydrogen bond network observed for AT (Fig. 3C). Clearly, in
AT, the active site is optimized to accommodate glycine. Its function
in creatine biosynthesis is assisted by the flexible 300-loop and helix
H9, resulting in a much more restricted active site. The kinetic and
crystallographic analysis of AT and of its deletion mutants suggested
that these key differences have led to (i) a strong increase of the
catalytic turnover, which is required for creatine biosynthesis, (ii) a
slightly diminished arginase side reactivity, and (iii) an increased
substrate specificity. Stability measurements showed that H9 may
increase the pH stability but has no function in temperature stability.