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J. Biol. Chem., Vol. 277, Issue 25, 22992-22999, June 21, 2002
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From the
Received for publication, March 8, 2002, and in revised form, April 3, 2002
We have probed the
structural/functional relationship of key residues in human placental
alkaline phosphatase (PLAP) and compared their properties with those of
the corresponding residues in Escherichia coli alkaline
phosphatase (ECAP). Mutations were introduced in wild-type PLAP,
i.e. [E429]PLAP, and in some instances also in [G429]PLAP, which displays properties characteristic of the human germ cell alkaline phosphatase isozyme. All active site metal ligands,
as well as residues in their vicinity, were substituted to alanines or
to the homologous residues present in ECAP. We found that mutations at
Zn2 or Mg sites had similar effects in PLAP and ECAP but that
the environment of the Zn1 ion in PLAP is less affected by
substitutions than that in ECAP. Substitutions of the Mg and Zn1
neighboring residues His-317 and His-153 increased kcat and increased Km when
compared with wild-type PLAP, contrary to what was predicted by the
reciprocal substitutions in ECAP. All mammalian alkaline phosphatases
(APs) have five cysteine residues (Cys-101, Cys-121, Cys-183, Cys-467,
and Cys-474) per subunit, not homologous to any of the four cysteines
in ECAP. By substituting each PLAP Cys by Ser, we found that disrupting the disulfide bond between Cys-121 and Cys-183 completely prevents the
formation of the active enzyme, whereas the carboxyl-terminally located
Cys-467-Cys-474 bond plays a lesser structural role. The substitution
of the free Cys-101 did not significantly affect the properties of the
enzyme. A distinguishing feature found in all mammalian APs, but not in
ECAP, is the Tyr-367 residue involved in subunit contact and located
close to the active site of the opposite subunit. We studied the A367
and F367 mutants of PLAP, as well as the corresponding double mutants
containing G429. The mutations led to a 2-fold decrease in
kcat, a significant decrease in heat stability,
and a significant disruption of inhibition by the uncompetitive
inhibitors L-Phe and L-Leu. Our
mutagenesis data, computer modeling, and docking predictions indicate
that this residue contributes to the formation of the hydrophobic
pocket that accommodates and stabilizes the side chain of the inhibitor during uncompetitive inhibition of mammalian APs.
Alkaline phosphatases
(APs1; EC 3.1.3.1) are a
family of dimeric metalloenzymes catalyzing the hydrolysis of
monoesters of phosphoric acid (1). APs also catalyze a
transphosphorylation reaction in the presence of large concentrations
of phosphate acceptors. APs occur widely in nature and are found in
many organisms from Escherichia coli to man. APs are
homodimeric enzymes, and each catalytic site contains three metal ions
(two Zn ions and one Mg ion) that are necessary for enzymatic activity
(2). Whereas the main features of the catalytic mechanism are conserved between mammalian APs and the E. coli enzyme, there are
important differences. Mammalian APs (a) have higher
specific activity and Km values, (b) have
a more alkaline pH optimum, (c) are inhibited by
L-amino acids and peptides through an uncompetitive mechanism, and (d) display lower heat stability. These
properties, however, differ noticeably within the group of mammalian AP
isozymes. Many of the isozyme-specific properties have been attributed
to a flexible loop region or "crown" domain of the molecule (3, 4).
Some mammalian APs, such as the human intestinal isozyme, are activated
by magnesium ions, whereas the human placental AP is more similar to
the E. coli enzyme in that its activity is not enhanced by
the addition of magnesium (1).
For many years, E. coli AP (ECAP) was the only source of
structural information on APs (2), but now the three-dimensional structure of the first mammalian AP, i.e. human placental
alkaline phosphatase (PLAP), has been solved (4). As had been predicted from sequence comparisons (5), the central core of PLAP, consisting of
an extended In the present paper, we embarked on a wide-range mutagenesis study on
structure-function relationships in mammalian APs, using the PLAP
structure as a paradigm. The aim was to pinpoint the features of PLAP
responsible for the specific properties of this AP isozyme, as well as
the properties of mammalian APs in general. The active site metal
ligand residues were mutated to alanines or to the analogous residues
in the structure of ECAP, and cysteines were mutated to serines.
Furthermore, Tyr-367 is conserved in all mammalian APs and occupies a
unique position in the PLAP structure, serving as a bridge from one
subunit in the dimer to the active site of another subunit. We
mutagenized the Tyr-367 residue to Ala and Phe. Mutations were studied
individually as well as in combination with the E429G substitution
because this change has been shown to significantly affect many of the enzymatic properties of PLAP by conferring germ cell alkaline phosphatase characteristics to the resulting mutant enzyme
(10-13).
The present analysis of structurally and catalytically important
residues in PLAP has identified critical positions serving a different
structural role in mammalian and bacterial alkaline phosphatases. We
have also defined the location of the hydrophobic pocket that
participates in stabilizing the side chains of uncompetitive inhibitors
in the immediate vicinity of the active site of mammalian alkaline phosphatases.
PLAP Mutagenesis
Site-directed mutagenesis was performed by PCR with mutated
oligonucleotide primers. The strategy involves the introduction of
restriction sites for enzymes that cut at a distance from their recognition sites (BsaI, BspMI,
Alw26I, and EarI) or the use of endogenous
restriction enzyme sites. Either the PLAP-FLAG/pcDNA3 or PLAP/pSVT7
plasmid was used as a template in PCR reactions. PCR products were
subcloned into a pCR2.1 or PCRII-TOPO cloning vector (Invitrogen), and
the mutations were confirmed by sequencing. The restriction fragments
were then cut and ligated with the fragments of PLAP-FLAG/pcDNA3
and pcDNA3 plasmid (Invitrogen). All final constructs were verified
by restriction enzyme analysis and sequencing. Plasmid DNA was prepared
by the alkaline lysis procedure. Sequencing was performed using
Sequenase according to the manufacturer's protocol (Amersham Biosciences).
Metal Ligand Mutations--
The sequences of the
oligonucleotide primers used for amplifying the site-directed
mutagenized fragments in the case of the active site metal ligands are
described in this section. The name of the primer (all are shown
5' to 3') is given first, followed by the sequence (positions that
denote the mutation are underlined): D42A,
GCG-GTC-TCC-TGG-GCG-CTG-GGA-TGG-G; 42
pSVT7 PLAP was used as the template in PCR reactions with
the following primer pairs: 1, T7 and 42
For other active site area mutations, PLAP-FLAG/pcDNA3 was used as
template in the following PCR reactions: 317-44, H317A and E429G( Cys Mutations--
To prepare the cysteine mutants, the
following PCR primers were used: F101,
ACA-GCC-GGT-CTC-TAC-CTG-AGC-GGG-GTC-AAG-GGC; R101, CTT-GAC-GGT-CTC-CAG-GTA-GGC-CGT-GGC-TGT; F121,
GCA-GCC-GGT-CTC-TTT-AAC-CAG-AGC-AAC-ACG-ACA-CGC; R121,
CGT-GTT-GGT-CTC-GTT-AAA-GCG-GGC-GGC-TGC; F183,
GCC-TCG-ACC-TGC-CAG-GAG-GGG-TCC-CAG-GAC; R183,
AGC-GAT-ACC-TGC-GCA-CCC-CTC-CTG-GCG-GGC; C467S,
GGG-GCG-CCA-GGT-CGC-AGG-CGG-TGT-AGG-GCT-CCA-GGG-AGG-CGG; C474S,
GGG-GCG-CCA-GGT-CGG-AGG-CGG-TGT-AGG-GCT-CCA-GGC-AGG-CGG; SSR, GGC-GGT-CTC-GGG-CTC-CAG-GGA-GGC-GGC-GAA; ALW,
GCC-GCC-CGT-CTC-GAG-CCC-TAC-ACC-GCC-TCC-GAC-CT; T7,
TAA-TAC-GAC-TCA-CTA-TAA-GGG; and SP6, ATT-TAG-GTG-AGA-CTA-TAG. The products of the PCR reactions (CYS1, F101 and
R183; CYS2, R101 and T7; CYS3, F121 and R183; CYS4, R121 and T7; CYS5,
R183 and T7; CYS6, F183 and SP6; CYS7, D357A and C467S; CYS8, D357A and
C474S; CYS9, D357A and SSR; and CYS10, ALW and SP6) were isolated and
subcloned into the pCR2.1 vector. The following restriction fragments
were cut and ligated with the fragments of PLAP-FLAG/pcDNA3 to make
the final constructs: CYS1/BsaI-BstEII and
CYS2/EcoRI-BsaI for the C101S mutant;
CYS3/BsaI-BstEII and
CYS4/EcoRI-BsaI for C121S;
CYS5/EcoRI-BspMI and
CYS6/BspMI-XbaI for C183S;
CYS7/PauI-KasI for C467S;
CYS8/PauI-KasI for C474S; and
CYS9/PauI-Alw26I and CYS10/Alw26I-XbaI for the double S467 + S474
mutation. All final constructs were verified by restriction enzyme
analysis and sequencing.
Mutations Involving Residue 367--
The following PCR primers
were used: Y367A(R), CGA AGA
TGG AGC TCC CTC
GCA GGG GGG CGC
CTC C; Y367F(R), CGA
AGA TGG AGC TCC CTC GCA GGG GGA
AGC CTC C; in PCR reactions. For
PCR reaction 367A/ we used primers H153A and
Y367A(R), whereas for reaction 367F/ the
primers H153A and Y367F(R) were used.
Restriction fragments were then cut and combined with fragments from
PLAP-FLAG/pCDNA3 to make the final constructs (for the [A367]PLAP
or [F367]PLAP mutants, BsmBI-SacI from 367A or
367F, respectively; for the [A367, G429]PLAP or [F367, G429]PLAP
mutants, SacI-XbaI from [E429G]PLAP and
BsmBI-SacI from A367 or F367, respectively).
Expression and Purification of FLAG-tagged Enzymes
PLAP-FLAG constructs were transfected into COS-1 cells for
transient expression by the DEAE-dextran- or calcium phosphate-mediated method. Three and 6 days after transfection, conditioned media were
collected and concentrated about 10 times by ultrafiltration on 50 Centricon columns (Amicon Inc., Beverly, MA). PLAP-FLAG proteins were
purified by affinity chromatography with anti-FLAG M2 antibody gel
(Sigma) according to the manufacturer's instructions.
Characterization of Recombinant APs
To measure relative specific activities, microtiter plates were
coated with 2 µg/ml M2 anti-FLAG monoclonal antibody (Sigma). After
the addition of recombinant APs, the activity of the bound enzymes was
measured as the change in absorbance at 405 nm over time at 37 °C
upon the addition of 20 mM
p-nitrophenylphosphate as substrate in 1.0 M
diethanolamine buffer (pH 9.8), 1 mM MgCl2, and
20 µM ZnCl2. PLAP-FLAG served as a reference
for each microtiter plate. The p-nitrophenol concentration
formed was calculated using an extinction coefficient of 10,080 liter × mole AP pH/activity profiles were done using 20 mM
p-nitrophenylphosphate as substrate in 50 mM
buffer with 1 mM MgCl2 and 20 µM ZnCl2; bis-tris propane for pH range 6.5-9.5, and
2-amino-2-methyl-1-propanol for pH range 9.5-11. The pH was checked
after dissolving the substrate. The 50 mM
3-(cyclohexylaminol)-1-propanesulfonic acid buffer proved to be
inhibitory to PLAP but not ECAP activity. The carboxyl-terminal FLAG
fusion protein of ECAP (Eastman Kodak Co.) was used for comparison. The
heat stability of PLAP mutants was studied by incubating the enzyme
samples in 1 M diethanolamine buffer, pH 9.8, containing 1 mM MgCl2 and 20 µM
ZnCl2. After a 10-min incubation at a given temperature,
the samples were placed on ice. Residual activities were then measured
in duplicate with 10 mM p-nitrophenylphosphate at pH 9.8 in the same buffer.
Fluorescence Labeling of PLAP
The cysteine-specific probe
4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F; Molecular
Probes Inc., Eugene, OR) was used to label wild-type PLAP (not modified
with FLAG peptide). Free ABD-F shows no fluorescence but can form
fluorescent adducts with protein thiol groups with an excitation
maximum at 390 nm and an emission maximum at about 500-520 nm. In a
modification reaction, 20-µl aliquots of 50 mM ABD-F
solution in Me2SO were added to 1-ml samples of PLAP
solution (0.4 µM/liter) in 0.1 M Tris-HCl
buffer, pH 7.6, either at nondenaturing conditions or in the presence
of denaturant (4 M guanidine hydrochloride), reducing agent
(1 mM tris-(2-carboxylethyl)phosphine; Molecular Probes Inc), or both. The samples were then incubated for 3 h at 37 °C while protected from light. Fluorescence spectra were measured with a
FluoroMax-2 fluorometer with excitation at 390 nm.
Computer Models and Docking Strategy
The binding of inhibitors L-Leu and
L-Phe to the PLAP molecule was modeled using the flexible
ligand docking program FlexX (14). The active site of PLAP was defined
as a sphere of 9 Å around the Zn1 atom. The phosphate ion present in
the enzyme structure was included in the receptor model. Ligands were
prepared in the SYBYL mol2 format, in energy-minimized form, with all
hydrogens added and formal charges assigned. The amino groups of
L-Leu and L-Phe were left unprotonated. The
manual "base fragment" selection (14) option was used, and either
the carboxylic, amino, or hydrophobic groups of the ligand were chosen
as the base fragment.
To pinpoint the residues important for catalysis and stability of
human PLAP, we constructed and characterized 23 individual site-directed mutants of PLAP (Fig. 1) as
well as 7 double mutations. Residues serving as ligands to
catalytically important metal ions, i.e. Asp-42, His-153,
Ser-155, Glu-311, Asp-316, His-320, Asp-357, His-358, His-360, and
His-432, were mutagenized into Ala. Two residues in the vicinity of the
Mg-binding site in PLAP that are not conserved in ECAP, i.e.
His-153 and His-317, were mutagenized into Ala or to Asp and Lys,
respectively. Some active site mutations were studied in the context of
both wild-type PLAP, i.e. [E429]PLAP, and [G429]PLAP
because this substitution has been shown to have profound influences on
the behavior of the enzyme by conferring upon it germ cell alkaline
phosphatase characteristics (11), i.e. [H153A, E429G],
[H317A, E429G], and [H319A, E429G]. Furthermore, the mammalian APs
have 5 cysteine residues/monomer in positions that are not homologous
to the 4 cysteines of ECAP (7), and we mutagenized each of these 5 cysteines of PLAP to Ser and also studied the [C467S, C474S] double
mutant. In addition, mutagenesis of the Tyr-367 residue, a signature
feature of mammalian APs, was also performed.
To simplify the recovery and purification of the recombinant PLAP
variants, the glycosylphosphatidylinositol anchoring sequence of
PLAP was replaced by the FLAG octapeptide, and all mutants were
expressed as secreted, epitope-tagged, enzymes. We had previously determined that the addition of the FLAG sequence does not interfere with the kinetic properties of the molecule (15). This strategy facilitated the production of large amounts of recombinant protein and
enabled the fast and efficient isolation of each mutant AP using
anti-FLAG affinity purification. Two substitutions resulted in mutant
enzymes that did not have any AP activity over background levels in the
culture media, i.e. C121S and C183S. We could not detect any
FLAG-tagged protein in the media for these mutants by Western blot
analysis, indicating that these mutations had severe adverse
consequences on protein structure, proper folding, and/or secretion.
Mutagenesis of Metal Ligands in PLAP--
Fig.
2 shows a detailed comparison of the
structure of the active site region of PLAP and ECAP including all the
metal ligands that were mutagenized in this study. When substituting
the Zn1 ligands in PLAP, i.e. Asp-316, His-320, and His-432,
two of the three mutants, i.e. [A316]PLAP and
[A432]PLAP, retained significant activity (Table
I). The kcat and
Km of [A316]PLAP showed a 2.8- and 2.25-fold
decrease relative to wt PLAP. Thus, the catalytic efficiency
(kcat/Km) of the [A316]PLAP
mutant remains comparable to that of wt PLAP. The
kcat of [A432]PLAP was also reduced 2.7-fold,
but its Km increased 3.7-fold for a resulting
5.8-fold reduction in catalytic efficiency. In contrast, the
introduction of the H320A mutation reduced the specific activity of
PLAP by >200-fold. Saturation of each of the mutants with
Zn2+ concentrations of up to 10 mM did not
result in any increase in activity. It should be noted that analogous
mutations in ECAP were reported to have very different consequences.
Notably, the D327A substitution in ECAP (analogous to Asp-316 in PLAP)
resulted in a 3000-fold decrease in kcat and a
2000-fold increase in Km for a 107-fold
decrease in catalytic efficiency that was not reversible by the
addition of Zn2+ (16). In contrast, the activity of the
H412A mutant in ECAP (analogous to H432A in PLAP) was responsive to 0.2 mM Zn2+, reaching kcat
and Km values only 2-fold lower that those of wt
ECAP (17). The H331A mutation (analogous to H320A in PLAP) has not been
studied in ECAP. These results indicate that there are significant
differences in the environment of Zn1 in the PLAP structure compared
with the ECAP structure and that substitutions of the Zn1 ligands are
better tolerated in PLAP than in ECAP. This may reflect the fact that
the top flexible loop, or crown domain, that harbors E429 in PLAP
appears to provide additional stabilization to the active site
environment, so that Zn2+ cannot easily diffuse in or out
of the PLAP molecule, as we have previously observed (18). Thus, even
though the state of coordination of Zn1 is affected by the mutations,
the Zn2+ ion remains in place and is able to function in
catalysis.
Alanine substitutions of the Zn2 ligands in PLAP, i.e.
Asp-42, Asp-357, and His-358, resulted in significant decreases in specific activity, ranging from >25-fold (D357A) to undetectable levels (H358A). None of these values changed in response to the addition of Zn2+. Whereas the Km of
[A42]PLAP nearly doubled, the Km of [A357]PLAP
decreased slightly. Thus, the catalytic efficiencies of [A42]PLAP and
[A357]PLAP were reduced by 130- and 16-fold, respectively. Whereas no
studies have been performed in ECAP on residues analogous to Asp-357
and His-358, the [A51]ECAP mutant, analogous to [A42]PLAP, was
shown to be >800-fold less active than the wt ECAP (19).
Ala-42 is a bidentate ligand, coordinating not only to Zn2 but also to
Mg. Alanine substitution of the other two Mg ligands, i.e.
Ser-155 and Glu-311, reduced the specific activity of PLAP ~100- and
200-fold, respectively. The Km for [A155]PLAP did
not change, but it increased about 4-fold for [A311]PLAP. A similar
pattern was seen for the corresponding E322A mutation in ECAP (20).
Interestingly, the S155T substitution hardly affects the activity of
the resulting mutant and even doubles its catalytic efficiency (Table
I).
Mutagenesis of Nonconserved Active Site Residues in
PLAP--
Whereas most of the AP active site residues are
perfectly conserved throughout evolution, some important differences
exist in the neighborhood of the Mg ion (Fig. 2). His-153 and His-317 in PLAP are homologous to Asp-153 and Lys-328, respectively, in ECAP.
The substitution of D153H and K328H in ECAP produced enzymes with
kinetic properties similar to those of mammalian APs. For example, the
D153H/K328H double ECAP mutant displayed a 5.6-fold higher
kcat and a 30-fold higher Km,
a decrease in heat stability, and a shift in pH optimum to alkaline pH
values (21). We constructed the reciprocal mutations, i.e.
H153D and H317K, in PLAP as well as the double mutation (H153D/H317K).
We also introduced a H153A and H317A mutation in both PLAP and
[G429]PLAP. The expectation was that by reverting to the residues
found in ECAP, one would confer ECAP-like properties to PLAP.
Surprisingly, however, no decrease in Km was
observed in any of the mutants. Instead, the Km
values consistently increased for all the mutants. The effects on
kcat were variable. There were no significant
changes in the pH dependence or heat stability of the mutants compared
with the wt PLAP (data not shown).
In the ECAP active site, the environment of the Mg ion is one of
octahedral coordination, including three amino acids and three water
molecules (2). These water molecules are further coordinated and
stabilized by other amino acid residues including Asp-153. The D153H
mutation in ECAP was shown to destabilize the octahedral Mg
coordination in favor of a tetrahedral one and resulted in an enzyme
that had reduced Mg2+ affinity and increased
Zn2+ affinity and was significantly activated by
Mg2+ (21). This is strongly reminiscent of the behavior of
the IAP isozyme (1) but not of human PLAP, which binds
Mg2+ tightly and in which the further addition of
Mg2+ does not increase activity. A possible explanation for
our results comes from the analysis of the structure of PLAP around the
Mg ion (see Fig. 2). In PLAP, His-153 and His-317 are positioned so
that they can serve the same purpose as the corresponding Asp and Lys
in ECAP, i.e. they are direct ligands to active site water molecules and indirect ligands to the Mg ion and the noncovalently bound phosphate group. We propose that His-153 and His-317 in PLAP are
already well positioned to stabilize these water-mediated interactions
and that introducing different residues at these positions would result
in a decrease in affinity for phosphate, thus increasing
Km and kcat rather than
decreasing these parameters. Because the IAP isozyme is more dependent
on Mg2+ activation, one can speculate that the structure of
IAP in the immediate environment of the Mg ion is more similar to and
can be better modeled by the structure of ECAP, rather than the
structure of PLAP. Upon mutagenizing H153A or H317A in PLAP, both
mutant enzymes displayed about a 2-fold increase in
kcat and about a 3-fold increase in
Km compared with wt PLAP. This can be explained by
the disruption of their water-mediated interactions with the phosphate
group (see Fig. 2) via the same water molecule. Disruption may lead to
an enzyme with smaller affinity for both substrate and product, thus
having higher kcat and Km values. Interestingly, by combining the H153A and H317A mutations with
the G429 mutation, we were able to engineer mutant enzymes with
increased catalytic efficiency when compared with wt PLAP but not when
compared with [G429]PLAP. The [A153, G429]PLAP and [A317,
E429G]PLAP mutants had Km values that were restored to those of wt PLAP while largely preserving the increase in
kcat. This is especially true of [A317,
G429]PLAP, in which both specific activity and catalytic efficiency
(kcat and
kcat/Km values) were
increased 2-fold compared with wt PLAP and 5-fold when compared with
[G429]PLAP.
Other Conserved Residues in the Active Site Area--
Two other
active site residues, His-360 and His-319, that are perfectly conserved
in mammalian APs were also investigated. In ECAP, His-372 is 3.8 Å away from Zn1 in the active site, and correspondingly, His-360 is
located within 4.4 Å from the Zn1 ion in PLAP. The side chain of
His-360 forms a hydrogen bond to the side chain of Asp-316, which is a
direct ligand to Zn1. The [A372]ECAP showed changes in catalytic
behavior, such as a 20% reduction in kcat and a
4-fold reduction in Km in the presence of a
phosphate acceptor (22). In contrast, [A360]PLAP displayed an
increase in specific activity 20% over that of wt PLAP and a 3.8-fold
increase in Km.
Judging from its three-dimensional positioning, residue His-319 should
mainly have a structural role, being involved in hydrogen bonds with
Thr-48 and Tyr-393. The H319A and H319A/E429G mutations, however,
displayed a 70-fold and 37-fold decrease in specific activity,
respectively, indicating that the interactions formed by His-319 are
important for maintaining an optimal conformation in the active site.
Hierarchical Significance of the Five PLAP Cysteines--
PLAP, as
well as all mammalian APs, has 5 cysteine residues/subunit in positions
101, 121, 183, 467, and 474 (Fig.
3A). They form two disulfide
bonds, Cys-121-Cys-183 and Cys-467-Cys-474, whereas the Cys-101 residue
remains in free form. We found that the cysteine recombinant PLAP
mutants C101S, C467S, C474S, and C467S/C474S were appropriately
expressed by transfected COS-1 cells and retained residual activity,
whereas the C121S and C183S mutants were degraded inside the cell. The
PLAP structure reveals that the sequence contained within the
Cys-121-Cys-183 disulfide bond (Fig. 3B) harbors three
important elements of secondary structure: (a) a stretch of
the central
The kcat and Km values for
the active mutants are summarized in Table I. No significant changes
were observed in the substrate affinity of these mutant enzymes as
compared with wt PLAP. In the case of [S101]PLAP, even the catalytic
efficiency was similar to that of wt PLAP. We examined the possibility
that Cys-101 would be available for covalent modification by the
fluorescent probe ABD-F. Only when wt PLAP was incubated with
guanidinium chloride did the Cys-101 side chain become available for
covalent modification (Fig.
4A). This confirms that
Cys-101 is not likely to play a role in regulating activity or
stability of the enzyme. In contrast, [S467]PLAP, [S474]PLAP, and
[S467, S474]PLAP enzymes all displayed kcat
values that were 2-fold lower than the wt PLAP value of 460 s Significance of the Conserved Y367--
An interesting structural
feature of PLAP, with no counterpart in the ECAP structure, is Tyr-367.
This residue is part of the subunit interface in the PLAP dimer, where
it protrudes from one subunit and is positioned within 5.6 Å of the
catalytic Zn1 ion in the active site of the other subunit (Fig.
5A). The HO
One of the specific properties of APs of higher organisms is their
ability to be inhibited stereo-specifically by L-amino acids and peptides through an uncompetitive mechanism (23). Our
previous studies have revealed that the nature of residue 429 has a
profound effect on the inhibition. The E429G, E429S, and E429H
substitutions, all naturally occurring substitutions (Fig. 1), had the
same effect of facilitating the access of the inhibitor to the active
site (10). Other residues located near 429 include Tyr-367 and His-317,
and their participation in the inhibition mechanism has not been
investigated previously. Most of the specific inhibitors of mammalian
APs contain a hydrophobic side chain, such as in L-Phe or
L-Leu. The nature of this side group affects the inhibition
properties, and it has been postulated that a hydrophobic pocket exists
to accommodate this part of the inhibitor molecule (24), although its
exact location remains unclear. We postulated that Tyr-367 might play a
role in stabilizing the inhibitor.
We mutated Tyr-367 to Phe as the closest possible structural analogue
of Tyr and to Ala. Both mutations were done in the context of wt PLAP
and [G429]PLAP. Residue 429 is situated close to Tyr-367, so it was
of interest to check the effect of the double substitutions. The
effects of the substitutions at Tyr-367 on the kinetic properties of
PLAP were very similar for all four recombinant mutants studied, i.e. [A367]PLAP, [F367]PLAP, [F367, G429]PLAP, and
[A367, G429]PLAP (Table I). The kcat values
were in the range of 39-45% of the wt PLAP value. The
Km values were not significantly changed from the
value of wt PLAP (0.35 mM). However, the Y367A and Y367F substitutions significantly compromised the heat stability of the
mutant PLAP enzymes (Fig. 6). In
addition, whereas [F367]PLAP, like wt PLAP, displayed a monophasic
inactivation curve, [A367]PLAP instead displayed a biphasic
inactivation mechanism. Interestingly, the [A367]PLAP mutant was more
stable than the [F367]PLAP enzyme after a prolonged incubation,
despite a higher initial rate of inactivation.
We then examined the inhibition properties of the mutants toward
L-Phe, L-Leu, and
L-(2-phenyl)-glycine (Fig.
7). Because all these enzyme variants had
comparable kinetic parameters, any observed changes in inhibition
properties should mainly reflect the changes in true binding constants
for the inhibitors studied (13). The Y367A mutation in PLAP was found
to have a very profound destabilizing effect on the inhibition by
L-Phe and L-Leu increasing their
Ki values by 17.5- and 12.7-fold, respectively (Table II). The Y367F substitution had
similar but less pronounced consequences, increasing
Ki values by 3.2- and 4-fold, respectively. These
results show that the side chain of Tyr-367 is crucial for the binding
of inhibitors to PLAP, most likely by providing a local binding area
for the hydrophobic side chains of Leu and Phe. In the double mutants,
[A367, G429]PLAP and [F367, G429]PLAP, the pattern of inhibition by
L-Leu and L-Phe was intermediate. The effect of
the Y367A substitution overweighed that of E429G, whereas the effect of
Y367F was completely rescued by the E429G mutation (Fig. 7). Inhibition
of [F367]PLAP and [A367]PLAP by D-Phe was even less
pronounced than the inhibition by L-Phe and was so low that
accurate determination of Ki was difficult (Ki > 90 mM) (data not shown). Whereas
the effect of the Y367A mutation was the most significant, the H317A
substitution also had a mild influence on inhibition by both
L-Phe and L-Leu, decreasing
Ki by 2.6- and 2.5-fold, respectively. The [A317,
G429]PLAP double mutant displayed a similar decrease in Ki compared with [G429]PLAP. We also studied the
inhibition of PLAP mutants by L-(2-phenyl)-glycine, which,
from a structural viewpoint, can be considered a "truncated" form
of L-Phe. The results (Table II) show that
L-(2-phenyl)-glycine is as potent an inhibitor of PLAP and
PLAP mutants as L-Phe and L-Leu. However, L-(2-phenyl)-glycine does not discriminate between the
Y367A and Y367F mutations, suggesting that its shorter hydrophobic side chain makes less contact with Tyr-367. Our results indicate that substitutions at positions 367 and 429 can act independently in determining inhibition properties of PLAP. It is clear that removal of
Glu-429 facilitates access of the inhibitor to the active site, whereas
removal of Tyr-367 eliminates stabilization of the inhibitor's positioning at the active site.
To better explain the inhibition results, we performed a docking
simulation of the binding of L-Phe and L-Leu to
wt PLAP. For that purpose, we used the program FlexX (14), which
performs flexible ligand docking by an incremental construction
mechanism. Ligands with nonprotonated amino groups were used, in
agreement with the conclusions from previous experimental studies on
the pH dependence of the inhibition (13). The enzyme active site was
defined as a spheric area 9 Å around the Zn1 atom. In agreement with
the discussion in Ref. 14, we found the results of docking simulation
to be sensitive to the selection of the "base fragment" of
the ligand, i.e. the first group for which the program tries to find the putative positions in the active site. Automatic selection of the base fragment led to a cluster of solutions located far from the
active site atoms, which was not relevant for the inhibition modeling.
Hence we used manual selection of the base fragment and tried all three
main fragments of ligand: carboxylic, amino, and hydrophobic group (the
phenyl ring for phenylalanine and a methyl group for leucine). After
manual selection of the carboxylic group as the base fragment, results
were similar to the automatic mode. When the amino group or the
hydrophobic moiety was chosen as the base fragment, the ligands docked
close to the active site Zn1, and a similar orientation of the
hydrophobic chain was obtained for both inhibitors. Furthermore, using
the hydrophobic group as the base fragment also positioned the
inhibitor at coordination distance to the active site Zn1, a feature
that we previously found to be important in the inhibition mechanism
(13). The results for L-Phe and L-Leu are
presented in Fig. 5, B and C, respectively,
where the conformations with the highest energy score are shown. As can
be seen, the predicted location of the hydrophobic group is similar for
L-Leu and L-Phe. The pocket accommodating this
hydrophobic group is formed by the side chains of Tyr-367, Phe-107,
Gln-108, and Asp-91. The polar groups of the ligands are found to
interact with Zn1 ion and with the side chains of Glu-429 and Tyr-367.
This docking model is consistent with our mutagenesis studies and
provides an explanation for the important role of Tyr-367 in the
inhibition of PLAP by amino acids. Whereas the hydrophobic phenyl ring
of Tyr-367 is important for the formation of the hydrophobic pocket in
PLAP, the HO
In conclusion, our analysis of the structure-function relationship of
residues conserved throughout evolution from the E. coli to
the mammalian enzymes has revealed largely conserved functions for
those residues that stabilize the active site Zn and Mg metal ions. The
nonhomologous disulfide bonds differ in their structural significance,
and the free cysteine in mammalian APs can be substituted without major
consequences to enzyme function. Significantly, we found that
nonconserved residues that contribute active site stabilization and
cross-talk between enzyme monomers also determine the heat stability
and uncompetitive inhibition properties of mammalian alkaline phosphatases.
*
This work was supported in part by Grants CA 42595 and HD
12889 from the National Institutes of Health and the Swedish Medical Research Council.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Contributed equally to this work.
**
To whom correspondence should be addressed: The Burnham Inst.,
10901 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-646-3130; Fax:
858-713-6272; E-mail: millan@burnham.org.
Published, JBC Papers in Press, April 5, 2002, DOI 10.1074/jbc.M202298200
The abbreviations used are:
AP, alkaline
phosphatase;
PLAP, placental alkaline phosphatase;
ECAP, Escherichia coli alkaline phosphatase;
TNAP, tissue-nonspecific alkaline phosphatase;
IAP, intestinal alkaline
phosphatase;
wt, wild-type;
ABD-F, 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole.
Function Assignment to Conserved Residues in Mammalian
Alkaline Phosphatases*
§,§¶,
, and¶**
Department of Medical Biosciences, Umeå
University, S-901 85 Umeå, Sweden, Center for Molecular and
Vascular Biology, University of Leuven, B-3000 Leuven, Belgium,
and ¶ The Burnham Institute, La Jolla Cancer Research Center,
La Jolla, California 92037
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-sheet and flanking 
-helices, is very similar to that
of ECAP. The same is true in the immediate vicinity of the three
catalytic ions. However, a number of distinctive features, including a
different positioning of the amino-terminal segment of the molecule and
the expanded top loop or "crown" domain, are now apparent (4). An
additional noncatalytic metal-binding site not present in ECAP was
uncovered, which appears to be occupied by calcium (4, 6). ECAP has
four cysteine residues that are all involved in disulfide bond
formation (7), whereas PLAP has five nonhomologous residues.
Furthermore, whereas ECAP is located in the periplasmic space of the
bacterium, PLAP is an ectoenzyme bound to the plasma membrane via a
glycosyl-phosphatidylinositol anchor (8, 9).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
,
ATG-GTC-TCG-CCC-AGG-AAG-ATG-ATG-AG; H153A,
CCG-GTC-TCG-TGC-AGG-CCG-CCT-CGC-CAG-CCG; H153D,
CCG-GTC-TCG-TGC-AGG-ACG-CCT-CGC-CAG-CCG; S155A,
CCG-GTC-TCG-TGC-AGC-ACG-CCG-CGC-CAG-CCG; S155T,
CCG-GTC-TCG-TGC-AGG-CCG-CCA-CGC-CAG-CCG 153155,
GCG-GTC-TCC-TGC-ACT-CGT-GTG-GTG-GT; E311A,
CCC-CGC-GGC-TTC-TTC-CTC-TTC-GTG-GCG-GGT-GGT; D316A,
AGG-GTC-TCC-GCA-TCG-CCC-ATG-GTC-ATC-AT; H317K,
AGG-GTC-TCC-GCA-TCG-ACA-AAG-GTC-ATC-AT; 316317, CCG-GTC-TCG-ATG-CGA-CCA-CCC-TCC-AC; H320A,
GCG-GTC-TCC-ATG-GTC-ATG-CTG-GAA-AGC-AGG; 320,
TCG-GTC-TCA-CCA-TGG-TCG-ATG-CGA-CC; D357A,
GCG-GTC-TCA-CTG-CCG-CCC-ACT-CCC-ACG-TC; H358A,
GCG-GTC-TCA-CTG-CCG-ACG-CCT-CCC-ACG-TC; H360A,
GCG-GTC-TCA-CTG-CCG-ACC-ACT-CCG-CCG-TC; 357358360, GAG-GTC-TCG-GCA-GTG-ACG-AGG-CTC-AG; H432A,
GCC-GCG-CGC-GAA-CAC-CGC-CAC-GTC-CTC-GCC-TGC-GGC-GGT-CTC-TTC; 317, ATG-GGT-CTC-GTC-GAT-GCG-ACC-ACC-CTC; PLAPFLAG,
TCA-CTT-GTC-ATC-GTC-GTC-CTT-GTA-GTC-GGT-GGT-GCC-GGC-GGG-GGG-CGC; H317A,
TTC-CTC-TTC-GTG-GAG-GGT-GGT-CGC-ATC-GAC-GCT-GGT-CAT; H319A, TTC-CTC-TTC-GTG-GAG-GGT-GGT-CGC-ATC-GAC-CAT-GGT-GCT-CAT-GA;
and E429G(),
GCC-GCG-CGC-GAA-CAC-CGC-CAC-GTC-CTC-GCC-TGC-GTG-GGT-CTC-ACC-GTC.
; 2, D42A and 153155; 3, H153A and 316317; 4, H153D and 316317; 5, S155A and 316317; 6, S155T and 316317; 7, E311A and H432A; 8, D316A and H432A; 9, H317K and H432A; 10, H320A and H432A; 11, D357A and H432A; 12, H358A
and H432A; 13, H360A and H432A; 14, S155A and 357358360; 15, S155A
and 320; and 16, D357A and PLAP-FLAG. PCR products were subcloned and
sequenced to verify sequence integrity, and then the following
fragments were isolated and combined with fragments from pSVT7 PLAP to
produce the indicated PLAP mutants in pSVT7: 1/HindIII-BsaI and 2/Bsa BamHI for
D42A; 2/BamHI-BsaI and
3/BsaI-SacI for H153A;
2/BamHI-BsaI and
4/BsaI-SacI for H153D;
2/BamHI-BsaI and
5/BsaI-SacI for S155A;
2/BamHI-BsaI and
6/BsaI-SacI for S155T; 7/SacII-SacI for E311A;
4,5,6/SacII-BsaI and
8/BsaI-SacI for D316A; 4,5,6/SacII-BsaI and
9/BsaI-SacI for H317K;
15/SacII-BsaI and 10/BsaI-SacI for H320A;
14/SacII-BsaI and
11/BsaI-SacI for D357A; 14/SacII-BsaI and
12/BsaI-SacI for H358A;
14/SacII-BsaI and
13/BsaI-SacI for H360A; and
11/EagI-BssHII for H432A.
HindIII-SacII from H153D and
SacII-BssHII from H317K were combined to
construct [D153, K317]PLAP. PLAP residues past 483 were replaced by
the 8-amino acid FLAG epitope by ligating wt
EcoRI-BssHII with
16/BssHII-Xba into pcDNA3 (Invitrogen).
HindIII-BssHII fragments for all the pSVT7 PLAP
mutants were subcloned with the
BssHII-XbaPLAP-FLAG fragment into pcDNA3 to
create constructs encoding carboxyl-terminal FLAG epitope-tagged
secreted APs.
);
319-44, H319A and E429G(); and 429, H357A and E429G(). After PCR
products were subcloned, the following restriction fragments were cut
and ligated together with PLAP-FLAG/pcDNA3 fragments to give final
constructs: 429/SacI-PauI for E429G;
317-44/EarI-SacI for H317A;
319-44/EarI-SacI for H319A;
317-44/EarI-SacI and
429/SacI-PauI for H317A and E429G; and
319-44/EarI-SacI and
429/SacI-PauI for the double H319A + E429G
mutation. The fragments XbaI-BsmBI from the H153A
final construct and BsmBI-XbaI from the E429G
final construct were combined to prepare [A153, G429]PLAP.
1 × cm1. To calculate
Km, substrate concentration was varied between 0.2 and 1.6 mM p-nitrophenylphosphate, and the
initial reaction rate was measured at 37 °C over a time interval of
5 min. Results were fit by nonlinear regression to the Michaelis-Menten equation using GraphPad Prism version 3.02 (GraphPad Software, San
Diego, CA).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (32K):
[in a new window]
Fig. 1.
Residues that have been mutagenized in this
study. The sequences compared include all mammalian APs
known to date and also include one chicken AP sequence in comparison to
the E. coli AP sequence. They are E. coli AP
(ECAP); chicken tissue-nonspecific AP (chTNAP); cat TNAP
(cTNAP); bovine TNAP (bTNAP); rat TNAP
(rTNAP), mouse TNAP (mTNAP); human TNAP
(hTNAP); bovine intestinal AP I isozyme (bIAP I);
bovine IAP II, III, and IV (bIAP II, bIAP
III, and bIAP IV); rat IAP I and II (rIAP
I and rIAP II); mouse IAP and embryonic AP
(mIAP and mEAP); human IAP (hIAP);
human germ cell AP (hGCAP); and human PLAP
(hPLAP). A colored box over the residue number
indicates that it is a ligand to the active site Mg
(yellow), Zn1 (green), Zn2 (purple),
or both Zn2 and Mg (blue). The disulfide bonds between
Cys-121-Cys-183 and Cys-467-Cys-474 are shown by a thin
line.
View larger version (35K):
[in a new window]
Fig. 2.
Comparison of the residues coordinating to
the active site metals in PLAP and ECAP. The top panels
focus on the environment of the Zn1 and Zn2 metal sites and their
ligands, whereas the bottom panels display the environment
of the Mg metal site and its ligands. Water molecules are shown as
red spheres. Green dotted lines denote
metal-ligand interactions and hydrogen bonds.
Kinetic parameters of PLAP mutants
-strand; (b) the Arg-166 residue known to be
crucial for catalysis (13); and (c) the ligands stabilizing
the fourth Ca ion. Thus, interfering with Cys-121-Cys-183 disulfide
bond formation appears to be incompatible with proper enzyme
folding.
View larger version (72K):
[in a new window]
Fig. 3.
Cysteine residues in PLAP.
A, overall view of the PLAP dimer molecule. Cysteines
are shown for one subunit in spacefill representation. The secondary
structure elements are shown for the other subunit with -helices in
purple and -strands in yellow.
B, the area of PLAP around cysteines 121, 183, and 101. The catalytically important Arg-166 and phosphate group in the active
site of the enzyme are also shown. The polypeptide segment between
Arg-166 and Cys-183 is shown in green. The figure was
prepared with the program VMD (25).
1. The double [S467, S474]PLAP mutant was marginally
more active than the [S467]PLAP or [S474]PLAP enzymes. The
stability of these PLAP mutations toward heat inactivation was tested
at 68 °C in the same buffer used for the activity measurements (Fig.
4B). All four cysteine mutants displayed stability similar
to that of wt PLAP, with [S467]PLAP and [S474]PLAP being slightly
less stable than the other enzymes. When compared with other PLAP
mutations known to affect the heat stability, such as the E429G
mutation, one can conclude that no significant stability changes
occurred with any of these cysteine mutations.
View larger version (18K):
[in a new window]
Fig. 4.
Properties of PLAP Cys mutants.
A, covalent modification of cysteine residues of
wild-type PLAP by the fluorescent probe ABD-F.
PLAP+den+red, modification of PLAP was carried out in
the presence of denaturant (4 M guanidine hydrochloride)
and reducing agent (1 mM tris-(2-carboxylethyl)phosphine);
PLAP+den, modification of PLAP was carried out
in the presence of denaturant (4 M guanidine
hydrochloride); PLAP, PLAP at native conditions.
Excitation was at 390 nm. B, heat stability of the PLAP
mutants involving cysteines. The enzyme samples were incubated at
68 °C in 1 M DEA buffer at pH 9.8 for the times
indicated. The mutants are [S101]PLAP (S101), [S467]PLAP
(467S), [S474]PLAP (474S), and [S467,
S474]PLAP (S467+S474). The corresponding heat inactivation
curves for wt PLAP and [G429]PLAP are shown for comparison.
group of Tyr-367 also forms a hydrogen bond to the peptide group nitrogen of His-432, a direct ligand to the Zn1 ion (4). The location
of Tyr-367 in the structure and the fact that this residue is perfectly
conserved in mammalian APs (Fig. 1) implicate this residue in an
important structural/functional role.
View larger version (33K):
[in a new window]
Fig. 5.
Location and environment of the Tyr-367
residue. A, top view of the entrance to the active
site, showing the position of the Tyr-367 residue from one subunit
(wireframe representation) in the immediate vicinity of the Glu-429
residue of the other subunit (spacefilling representation).
B, computer modeling of L-Phe binding to
PLAP. C, computer modeling of L-Leu binding
to PLAP. The program FlexX (14) was used for the docking predictions.
The conformations of the ligands with the best energy score are shown.
The program Pymol (pymol.sourceforge.net) was used to generate the
figures.
View larger version (16K):
[in a new window]
Fig. 6.
Heat stability of Tyr-367 mutants.
Samples of wt PLAP, [A367]PLAP, and [F367]PLAP were incubated at
68 °C in 1 M DEA buffer at pH 9.8 for the times
indicated.
View larger version (29K):
[in a new window]
Fig. 7.
Inhibition curves of PLAP mutants by
L-Phe, L-Leu, and
L-(2-phenyl)-glycine. The graphs plot residual
activity versus inhibitor concentrations. The mutants tested
were wt PLAP, [G429]PLAP (E429G), [A367]PLAP (Y367A), [F367]PLAP
(Y367F), [A317]PLAP (H317A), [A367, G429]PLAP (Y367A/E429G),
[F367, G429]PLAP (Y367F/E429G), and [A317, G429]PLAP
(H317A/E429G).
Inhibition properties of single and double mutants of PLAP
group of Tyr-367 might help maintain the
correct orientation of phenyl ring via a hydrogen bond to the backbone
nitrogen of His-432. This HO group may additionally be
involved in a hydrogen bond with the polar groups of the ligand, as was
found in some of the docking solutions.
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1.
McComb, R. B.,
Bowers, G. N.,
and Posen, S.
(1979)
Alkaline Phosphatases
, Plenum Press, New York
2.
Kim, E. E.,
and Wyckoff, H. W.
(1991)
J. Mol. Biol.
218,
449-464[CrossRef][Medline]
[Order article via Infotrieve]
3.
Bossi, M.,
Hoylaerts, M. F.,
and Millán, J. L.
(1993)
J. Biol. Chem.
268,
25409-25416 4.
Le Du, M. H.,
Stigbrand, T.,
Taussig, M. J.,
Menez, A.,
and Stura, E. A.
(2001)
J. Biol. Chem.
276,
9158-9165 5.
Kim, E. E.,
and Wyckoff, H. W.
(1990)
Clin. Chim. Acta
186,
175-187[CrossRef][Medline]
[Order article via Infotrieve]
6.
Mornet, E.,
Stura, E.,
Lia-Baldini, A. S.,
Stigbrand, T.,
Menez, A.,
and Le Du, M. H.
(2001)
J. Biol. Chem.
276,
31171-31178 7.
Sone, M.,
Kishigami, S.,
Yoshihisa, T.,
and Ito, K.
(1997)
J. Biol. Chem.
272,
6174-6178 8.
Micanovic, R.,
Brink, B. L.,
Gerber, L.,
Pan, Y.-C.,
Hulmes, J. D.,
and Udenfriend, S.
(1988)
Proc. Natl. Acad. Sci. U. S. A.
85,
1398-1402 9.
Ogata, S.,
Hayashi, Y.,
Takami, N.,
and Ikehara, Y.
(1988)
J. Biol. Chem.
263,
10489-10494 10.
Hoylaerts, M. F.,
and Millán, J. L.
(1991)
Eur. J. Biochem.
202,
605-616[Medline]
[Order article via Infotrieve]
11.
Hummer, C.,
and Millán, J. L.
(1991)
Biochem. J.
274,
91-95
12.
Watanabe, T.,
Wada, N.,
Kim, E. E.,
Wyckoff, H. W.,
and Chou, J. Y.
(1991)
J. Biol. Chem.
266,
21174-21178 13.
Hoylaerts, M. F.,
Manes, T.,
and Millán, J. L.
(1992)
Biochem. J.
286,
23-30
14.
Rarey, M.,
Kramer, B.,
Lengauer, T.,
and Klebe, G.
(1996)
J. Mol. Biol.
261,
470-489[CrossRef][Medline]
[Order article via Infotrieve]
15.
Di Mauro, S., Manes, T., Hessle, H., Kozlenkov, A., Pizauro, J. M., Hoylaerts, M. F., and Millán, J. L. (2002)
J. Bone Miner. Res., in press
16.
Xu, X.,
and Kantrowitz, E. R.
(1992)
J. Biol. Chem.
267,
16244-16251 17.
Ma, L.,
and Kantrowitz, E. R.
(1994)
J. Biol. Chem.
269,
31614-31619 18.
Hoylaerts, M. F.,
Manes, T.,
and Millán, J. L.
(1997)
J. Biol. Chem.
272,
22781-22787 19.
Tibbitts, T. T.,
Murphy, J. E.,
and Kantrowitz, E. R.
(1996)
J. Mol. Biol.
257,
700-715[CrossRef][Medline]
[Order article via Infotrieve]
20.
Xu, X.,
and Kantrowitz, E. R.
(1993)
Biochemistry
32,
10683-10691[CrossRef][Medline]
[Order article via Infotrieve]
21.
Murphy, J. E.,
Tibbitts, T. T.,
and Kantrowitz, E. R.
(1995)
J. Mol. Biol.
253,
604-617[CrossRef][Medline]
[Order article via Infotrieve]
22.
Xu, X.,
Qin, X. Q.,
and Kantrowitz, E. R.
(1994)
Biochemistry
33,
2279-2284[CrossRef][Medline]
[Order article via Infotrieve]
23.
Fishman, W. H.,
and Sie, H.-G.
(1971)
Enzymologia
41,
140-167
24.
Ramasamy, I.,
and Butterworth, P. J.
(1975)
Biochim. Biophys. Acta
384,
146-158[Medline]
[Order article via Infotrieve]
25.
Humphrey, W.,
Dalke, A.,
and Schulten, K.
(1996)
J. Mol. Graphics
14,
33-38[CrossRef][Medline]
[Order article via Infotrieve]
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