(Received for publication, January 19, 1996; and in revised form, February 15, 1996)
From the
The crucial role of two reactive arginyl residues within the
substrate binding domain of human Type I D-myo-inositol 1,4,5-trisphosphate
(Ins(1,4,5)P) 5-phosphatase has been investigated by
chemical modification and site-directed mutagenesis. Chemical
modification of the enzyme by phenylglyoxal is accompanied by
irreversible inhibition of enzymic activity. Our studies demonstrate
that phenylglyoxal forms an enzyme-inhibitor complex and that the
modification reaction is prevented in the presence of either
Ins(1,4,5)P
, D-myo-inositol
1,3,4,5-tetrakisphosphate (Ins(1, 3,4,5)P
) or
2,3-bisphosphoglycerate (2,3-BPG). Direct
[
H]Ins(1,4,5)P
binding to the
covalently modified enzyme is dramatically reduced. The stoichiometry
of labeling with
C-labeled phenylglyoxal is shown to be
2.1 mol of phenylglyoxal incorporated per mol of enzyme. A single
[
C]phenylglyoxal-modified peptide is isolated
following
-chymotrypsin proteolysis of the radiolabeled
Ins(1,4,5)P
5-phosphatase and reverse-phase high
performance liquid chromatography (HPLC). The peptide sequence (i.e. M-N-T-R-C-P-A-W-C-D-R-I-L) corresponds to amino acids
340-352 of Ins(1,4,5)P
5-phosphatase. An estimate of
the radioactivity of the different phenylthiohydantoin amino acid
derivatives shows the modified amino acids to be Arg-343 and Arg-350.
Furthermore, two mutant enzymes were obtained by site-directed
mutagenesis of the two arginyl residues to alanine, and both mutant
enzymes have identical UV circular dichroism (CD) spectra. The two
mutants (i.e. R343A and R350A) show increased K
values for Ins(1,4,5)P
(10-
and 15-fold, respectively) resulting in a dramatic loss in enzymic
activity. In conclusion, we have directly identified two reactive
arginyl residues as part of the active site of Ins(1,4,5)P
5-phosphatase. These results point out the crucial role for substrate
recognition of a 10 amino acids-long sequence segment which is
conserved among the primary structure of inositol and
phosphatidylinositol polyphosphate 5-phosphatases.
In a wide variety of cell types D-myo-inositol
1,4,5-trisphosphate (Ins(1,4,5)P) (
)and
1,2-diacylglycerol are generated from phosphatidylinositol
4,5-bisphosphate (PtdIns(4,5)P
) by receptor-mediated
activation of phospholipase C (for review, see (1) and (2) ). Ins(1,4,5)P
mobilizes intracellular calcium
from internal stores generating calcium signals to control many
cellular processes: smooth muscle contraction, secretion, sensory
perception, neuronal signaling, and cell growth(2) .
Ins(1,4,5)P
can be dephosphorylated by a 5-phosphatase to
produce Ins(1,4)P
and phosphorylated by a 3-kinase to
produce Ins(1,3,4,5)P
(3, 4, 5) (for review, see Refs. 2, 6,
and 7). Some evidence supports a role for Ins(1,3,4,5)P
in
the regulation of intracellular free calcium concentration in concert
with Ins(1,4,5)P
(for review, see (7) and (8) ). Recently, a specific Ins(1,3,4,5)P
-binding
protein has been isolated and identified as a member of the GAP1
family, suggesting a connection between phospholipase C-derived signals
and a proliferative cascade involving Ras(9) .
A 75-kDa
inositol polyphosphate 5-phosphatase was initially identified in human
platelet lysates(10) . cDNAs encoding the enzyme have been
isolated from human cDNA libraries(11, 12) . When
expressed in COS cells, it shows Ins(1,4,5)P,
Ins(1,3,4,5)P
, PtdIns(4,5)P
, and
PtdIns(3,4,5)P
5-phosphatase
activities(13, 14) . A protein identified due to its
deficiency in the Lowe's oculocerebrorenal syndrome has been
shown to be homologuous to the 75-kDa inositol polyphosphate
5-phosphatase(15) . Expression of a truncated form of the
protein demonstrates Ins(1,4,5)P
,
Ins(1,3,4,5)P
, and PtdIns(4,5)P
5-phosphatase
activities(13) . In brain, Type I 43-kDa Ins(1,4,5)P
5-phosphatase is the major enzyme hydrolyzing the
calcium-mobilizing second messenger Ins(1,4,5)P
. It
hydrolyzes both Ins(1,4,5)P
and Ins(1,3,4,5)P
with higher affinity for Ins(1,3,4,5)P
but lower
velocity(16) . PtdIns(4,5)P
and
PtdIns(3,4,5)P
are not
substrates(14, 17) . cDNAs encoding Type I
Ins(1,4,5)P
5-phosphatase have been isolated from several
dog and human cDNAs libraries(18, 19, 20) .
Arginyl residues are known to act as anionic binding sites in
proteins and may thus assist in the binding of substrates or enzyme
catalysis. Covalent and irreversible modification with amino acid
specific reagents has been used successfully to identify lysyl or
arginyl residues in the substrate binding domain in many enzymes, such
as tyrocidine synthetase 1(21) ,
Ca/ATPase(22) ,
6-phosphofructo-2-kinase(23) , and Ins(1,4,5)P
3-kinase (24) . In addition, two arginyl residues were
shown using site-directed mutagenesis to be critical to bind the C-2
phospho group of fructose 2,6-bisphosphate in rat liver fructose
2,6-bisphosphatase(25) . We therefore investigated the
possibility that active site arginines may play such a role in
substrate binding for Type I Ins(1,4,5)P
5-phosphatase.
In this study, modification and inactivation of human Type I
Ins(1,4,5)P 5-phosphatase by phenylglyoxal, an
arginine-specific chemical modification reagent (see (26) ),
was shown to be prevented by Ins(1,4,5)P
. We identified two
essential arginines, i.e. Arg-343 and Arg-350, taking part of
the sequence segment R-X-P-A-W-C-D-R-I-L. This segment appears
to be crucial for enzymic activity, especially for substrate binding.
The data have been confirmed by site-directed mutagenesis of both
arginyl residues. This represents the first direct identification of an
inositol polyphosphate 5-phosphatase active site peptide.
Figure 1:
Time course of inactivation of
Ins(1,4,5)P 5-phosphatase by phenylglyoxal. Purified native (A) and expressed (B) Ins(1,4,5)P
5-phosphatase were incubated at 23 °C (pH 7.4) in the
presence of indicated concentrations of phenylglyoxal. Aliquots were
removed at indicated times and assayed for residual activity as
described under ``Experimental Procedures.'' Rate constants (k) were calculated from plots of the negative natural
logarithm of residual activity versus time. The results are
from one representative experiment out of three. C, The
reciprocal of k was plotted against the reciprocal of the
concentration of phenylglyoxal.
The first order reaction may be described by where (V/V) is the residual enzymic activity, k is the observed first order rate constant of inactivation, and t is the time of reaction with phenylglyoxal(33) .
A steady state treatment of the reaction described in yields (34) where K = k
/k
.
The linearity of a secondary plot of (1/k) versus (1/[phenylglyoxal]) using the data from the primary plot of Fig. 1indicated that phenylglyoxal binding takes place through the two-step mechanism of interaction described by (Fig. 1C).
Figure 2:
Protection against phenylglyoxal
inactivation by Ins(1,4,5)P. Ins(1,4,5)P
5-phosphatase was incubated at pH 7.4 and 23 °C in the
presence 20 mM phenylglyoxal and different concentrations of
Ins(1,4,5)P
(0-100 µM). Aliquots were
withdrawn at time intervals and assayed for enzymic activity. The
inactivation rate constants were calculated from these data as
described under ``Results.''
Figure 3:
Direct
[H]Ins(1,4,5)P
binding to
Ins(1,4,5)P
5-phosphatase before and after chemical
modification by phenylglyoxal. The enzyme was covalently modified in
the presence of 20 mM phenylglyoxal during 10 min as described
under ``Experimental Procedures.'' Unmodified (open
triangles) and modified (filled squares) enzyme have been
used in the [
H]Ins(1,4,5)P
binding
assay. [
H]Ins(1,4,5)P
(40,000 cpm)
was at 8 nM. Displacement was conducted with added unlabeled
Ins(1,4,5)P
(0-75 µM); 100% corresponds
to approximately 4,350 cpm of bound
[
H]Ins(1,4,5)P
.
Figure 4:
Relationship between incorporation of
phenylglyoxal protected by Ins(1,4,5)P and enzyme
inactivation. A, Ins(1,4,5)P
5-phosphatase was
incubated at 23 °C and pH 7.4 for various times (0-30 min)
with 20 mM [
C]phenylglyoxal in the
absence (filled squares) or presence (open triangles)
of 70 µM Ins(1,4,5)P
. Incorporation of
radioactive phenylglyoxal was estimated as described under
``Experimental Procedures.'' B, Ins(1,4,5)P
5-phosphatase was incubated as described in A in the
absence of any substrate. Residual activity and stoichiometry of
labeling were determined as described
previously.
Figure 5:
Reverse-phase HPLC profile of labeled
Ins(1,4,5)P 5-phosphatase digested by
-chymotrypsin. A, Ins(1,4,5)P
5-phosphatase was incubated in the
presence of 10 mM [
C]phenylglyoxal at
23 °C (pH 7.4) for 30 min and digested by
-chymotrypsin.
Resulting peptides were separated on a C
reverse-phase
HPLC column using a gradient of acetonitrile in 0.1% heptafluorobutyric
acid. An arrow head indicates the position of the major radioactive
peak. B, ordinate represents the radioactivity
detected in each peak of profile shown in A. C,
Ins(1,4,5)P
5-phosphatase was incubated as described in A except that labeling was performed in the presence of 70
µM Ins(1,4,5)P
. Ordinate represents
the radioactivity detected in each peak of the HPLC profile, which was
identical to the profile shown in A as mentioned under
``Results.''
Figure 6: Purification of the labeled peptide by reverse-phase HPLC. A, profile after separation of the peptide indicated by an arrow head in HPLC profile shown in Fig. 5A (after labeling in the absence of any substrate), using a gradient of acetonitrile in 0.1% trifluoroacetic acid. An arrowhead indicates the position of the radioactive peak. B, ordinate represents the radioactivity detected in each peak of the profile.
Figure 7:
Amino acid sequence determination of the C-radiolabeled peptide. Histogram showing the
radioactivity released during each cycle of Edman degradation of the
labeled peptide. The lower lines indicate the experimental sequence (13
amino acids) which corresponds to residues 340-352 of human brain
Type I Ins(1,4,5)P
5-phosphatase(12) , and the
yield quantified at each cycle by the protein sequenator. Dash
(-) indicates no amino acid detectable (i.e. phenylthiohydantoin-cysteine).
Figure 8:
Circular dichroism spectra of wild-type,
R343A, and R350A InsP 5-phosphatase. Millidegrees of CD
signal is plotted versus wavelength for the wild-type and the
two mutants R343A and R350A. Filled squares, wild-type; open circles, R343A; open triangles, R350A. The
signals are corrected to a comparable A
for each
enzyme preparation. Secondary structure estimates consistent with these
data are helix, 27.0 ± 0.7%,
, 25.8 ± 2.7%, turn,
28.4 ± 1.3%, and random, 18.7 ±
1.5%.
In this study, we aimed to identify active site residues
interacting with the substrate for human Type I InsP 5-phosphatase. This was investigated in a first step without any
assumption concerning the localization of reactive arginyl residues by
covalent chemical modification with phenylglyoxal, and in a second step
by site-directed mutagenesis of the previously identified arginyl
residues.
Inactivation kinetics of the enzyme by
phenylglyoxal-induced chemical modification are identical for native
and expressed protein. Our results indicate that the amount of
phenylglyoxal labeling paralleled the loss in enzyme activity and that
the labeling involves two residues. The peptide mapping of the protein,
which had been labeled with radioactive phenylglyoxal, enabled us to
find a peptide which was preferentially labeled in the absence of
substrate. We have identified two reactive arginyl residues within the
active site of Ins(1,4,5)P 5-phosphatase, i.e. Arg-343 and Arg-350 (Fig. 9). The identification of both
reactive amino acids is supported by two lines of evidence. First, the
phenylthiohydantoin residues corresponding to the cycles where the
modified arginines should elute contained radioactivity. Second, the
yield of unmodified arginine in these cycles was particularly low. The
covalent modification results in both an inactivation of the enzyme and
a drastic decrease in Ins(1,4,5)P
binding. This strongly
suggests that the Ins(1,4,5)P
binding domain of human Type
I Ins(1,4,5)P
5-phosphatase is the target of the chemical
modification. The role of Arg-343 and Arg-350 in substrate binding to
the enzyme is also supported by the catalytic properties of the two
mutants where Arg-343 and Arg-350 have been mutated to alanine. The
similarity in the induction yields and circular dichroism spectra
between the mutant and the wild-type enzymes indicate that mutation of
these arginyl residues did not affect the gross secondary structure of
the enzymes. However, the R343A and R350A mutants display a higher K
for Ins(1,4,5)P
as compared to the
wild-type enzyme (10- and 15-fold, respectively). The dramatic decrease
in affinity for Ins(1,4,5)P
for both the R343A and R350A
mutant enzymes indicates that both Arg-343 and Arg-350 are involved in
binding Ins(1,4,5)P
. Although both mutants have decreased
affinities for substrate, they exhibit distinct effects on the V
for Ins(1,4,5)P
5-phosphatase. The
more important change in V
for R343A mutant
enzyme suggest that Arg-343 may also be involved in Ins(1,4,5)P
5-phosphatase catalysis. Taking together, our data clearly
indicate for the first time that Arg-343 and Arg-350 are two reactive
residues involved in Ins(1,4,5)P
binding by human Type I
Ins(1,4,5)P
5-phosphatase.
Figure 9:
Aligment of human inositol and
phosphatidylinositol polyphosphate 5-phosphatase amino acid sequences
and location of [C]phenylglyoxal-labeled arginyl
residues in human Type I Ins(1,4,5)P
5-phosphatase. 43
kDa 5-phosphatase refers to the amino acid sequence of human Type
I Ins(1,4,5)P
5-phosphatase(19) . 75 kDa
5-phosphatase refers to the amino acid sequence of human inositol
polyphosphate 5-phosphatase(11) . OCRL protein refers
to the amino acid sequence of human Lowe's syndrome
PtdIns(4,5)P
5-phosphatase gene open reading
frame(15) . The 10-amino acid-long sequence segment is well
conserved between inositol and phosphatidylinositol polyphosphate
5-phosphatase sequences. It includes R-343 and R-350 (double
underlined) which were covalently labeled with
[
C]phenylglyoxal. Amino acids that are conserved
between the three sequences are indicated in bold. Amino acids
are represented in the one-letter code.
A lysine-rich
Ins(1,3,4,5)P-binding motif
(R/K-R/K-T-K-X-R/K-R/K-K-T) has been identified in
synaptotagmin II(35) . Moreover, a polybasic motif has also
been proposed to be necessary for PtdIns(4,5)P
binding
(R/K-X-X-X-X-K-X-R/K-R/K)
and allosteric Ins(1,4,5)P
binding to actin-binding
proteins and phospholipase C-
,
respectively(36, 37) . The pleckstrin homology domain
is a noncatalytic protein module of approximately 120 amino acids which
present the ability to bind PtdIns(4,5)P
and
Ins(1,4,5)P
(38) . The specific function of the
pleckstrin homology domain has not yet been elucidated. However, none
of those motives are found in the amino acid sequence of Type I
Ins(1,4,5)P
5-phosphatase, nor in other Ins(1,4,5)P
binding proteins such as Ins(1,4,5)P
3-kinases and
Ins(1,4,5)P
receptor/calcium channels. The primary function
of the Ins(1,4,5)P
binding domain is supposed to anchor the
inositol cycle and the three phosphates in position 1, 4, and 5. This
probably occurs through locking the inositol cycle in a hydrophobic
pocket and binding the phosphates with positively charged residues.
Arg-343 and Arg-350 of Type I Ins(1,4,5)P
5-phosphatase
could play an active role in this last interaction. Indeed, the use of
arginine to stabilize the binding of the phosphate moieties of the
substrate is quite a common occurrence in proteins for which the
three-dimensional structure is known, such as 6-phosphofructo-1-kinase (39) , glycogen phosphorylase b(40) , and
fructose 1,6-bisphosphatase. An arginyl residue of fructose
1,6-bisphosphatase belongs to the active site where the 6-phosphate
group is bound: this basic residue is conserved in inositol
monophosphatase sequence, which shares a very similar secondary
structure topology with fructose 1,6-bisphosphatase(41) . Since
Ins(1,4,5)P
and Ins(1,3,4,5)P
have a net charge
of -3 and -4, respectively, both molecules can interact
with a cluster of basic amino acids, at least with Arg-343 and Arg-350.
Results of the present study indicate that Arg-343 and Arg-350 in Type
I Ins(1,4,5)P
5-phosphatase take part of an active peptide
which may form a core where electrostatic and weak group (inositol
ring)-specific interactions take place.
Molecular cloning and
structural studies revealed that Ins(1,4,5)P 3-kinase and
PtdIns 3-kinase (for review, see (42) ) present strong
substrate specificities and do not share any sequence homology.
Although primary structure corresponding to inositol and
phosphatidylinositol polyphosphate 5-phosphatases present little amino
acid identity, it is intriguing to note that both reactive arginyl
residues Arg-343 and Arg-350 (in human Type I Ins(1,4,5)P
5-phosphatase sequence) identified in this study take part of a
COOH-terminal sequence segment, i.e.
R-C-P-A-W-C-D-R-I-L
(active site arginines are indicated in bold), which is well
conserved between sequences corresponding to inositol and
phosphatidylinositol polyphosphate 5-phosphatases (Fig. 9). It
would be of interest to investigate the effect of phenylglyoxal on the
enzymic activity of other phosphatases involved in the
dephosphorylation of inositol and phosphatidylinositol polyphosphate
molecules since these enzymes may present structural similarities.
These 5-phosphatases bind and hydrolyze the C-5 phospho group of
substrates, i.e. Ins(1,4,5)P
,
Ins(1,3,4,5)P
, PtdIns(4,5)P
, or
PtdIns(3,4,5)P
, each presenting an inositol cycle with at
least two phospho groups in position 4 and 5 of the inositol ring. We
suppose that other amino acid regions in Type I Ins(1,4,5)P
5-phosphatase, probably also located in the carboxyl-terminal
half of the protein, may serve together with residues 343-352 to
form a more precise conformation which is critical for the specific
binding of Ins(1,4,5)P
and Ins(1,3,4,5)P
.
However, the presence of a conserved active peptide within the primary
structure of inositol and phosphatidylinositol polyphosphate
5-phosphatases suggests that this family of enzymes present common
features in the mechanism of substrate binding and/or catalysis. This
10-amino acid-long peptide, which is critical for substrate binding in
Type I Ins(1,4,5)P
5-phosphatase, could represent a
diagnostic motif for this family of 5-phosphatases.