(Received for publication, November 11, 1996, and in revised form, January 14, 1997)
From the Department of Molecular Pharmacology, Albert
Einstein College of Medicine, Bronx, New York 10461 and the
§ Hubrecht Laboratory, Netherlands Institute for
Developmental Biology, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands
The catalytic activity and substrate specificity
of protein-tyrosine phosphatase (PTP
) is primarily controlled by
the membrane proximal catalytic domain (D1). The membrane distal (D2)
domain of PTP
by itself is a genuine PTPase, possessing catalytic
activity comparable to that of D1 using aryl phosphates as
substrates. Surprisingly, kcat and
kcat/Km for the
D2-catalyzed hydrolysis of phosphotyrosine-containing peptides
are several orders of magnitude reduced in comparison with those
of D1. Substitution of the putative general acid/base Glu-690 in D2 by
an Asp, which is invariably found in the WPD motifs in all cytoplasmic
PTPases and all the D1 domains of receptor-like PTPases, only
increases the kcat for D2 by 4-fold. Thus the
much reduced D2 activity toward peptide substrates may be due to
structural differences in the active sites other than the general
acid/base. Alternatively, the D2 domain may have a functional active
site with a highly stringent substrate specificity. PTP
display
modest peptide substrate selectivity and are sensitive to charges
adjacent to phosphotyrosine. In the sequence context of DADEpYLIPQQG
(where pY stands for phosphotyrosine), the minimal sizes recognized by
PTP
are either ADEpYLI or DADEpY-NH2.
Protein-tyrosine phosphatases (PTPases)1 catalyze the hydrolysis of phosphoryl groups on tyrosine residues in proteins that are introduced by protein-tyrosine kinases. A tightly balanced phosphatase and kinase activity is required for proper cellular functions (1, 2). PTPases constitute a growing family (>70 members) of enzymes that can be structurally categorized into two major groups: receptor-like (transmembrane) and non-receptor (intracellular) PTPases (3). Although many PTPases are proteins of greater than 400 amino acids, their catalytic domains are usually contained within a span of 250 residues referred to as the PTPase domain. This domain is the only structural element that has amino acid sequence identity among all PTPases from bacteria to mammals (4). The hallmark that defines the PTPase family is the active site sequence (H/V)C(X)5R(S/T), called the PTPase signature motif in the catalytic domain (5, 6). The receptor-like PTPases, exemplified by the leukocyte phosphatase CD45, generally have an extracellular domain, a single transmembrane region, and cytoplasmic PTPase domain(s). The intracellular PTPases, exemplified by PTP1B, contain a single catalytic domain and various amino- or carboxyl-terminal extensions including SH2 domains that may have targeting or regulatory functions.
The cytoplasmic segment of many receptor-like PTPases have two tandem
PTPase domains: D1, which is proximal to the membrane, and D2, which is
distal to the membrane. The existence of homologous PTPase domains in
the receptor-like PTPases raises the interesting possibility of
differential functions or regulations of the two domains.
However, the significance of the repeated PTPase domain in the
receptor-like PTPases is not clear. One important question is
whether both PTPase domains in the receptor-like phosphatases are
catalytically active? There is evidence that suggest that D2 is
catalytically inactive and may only play, if any, a regulatory role.
This is supported by the following observations. First, there are
receptor-like PTPases that lack either the essential Cys residue within
the PTPase signature motif in D2 (HPTP and RPTP
) (7, 8) or the
entire D2 domain (PTP
and DPTP10D) (7, 9-11). Second, replacement
of the Cys residue in the signature motif of D1 in LAR and CD45
abolishes the phosphatase activity while the same substitution in the
signature motif in D2 has little or no effect on PTPase activity (12,
13). Third, recombinant D1 of LAR or LCA (13-15) exhibits phosphatase
activity that are identical to the wild-type double domain PTPase,
whereas constructs for D2 alone show no measurable activity (13, 14).
Last, inactivation of D1 in PTP
(16) or CD45 (17) results in loss of
biological activity of these two PTPases, indicating that catalytic
activity of D1 is essential for the function of these PTPases.
On the other hand, there are reports that indicate that in some
receptor-like PTPases the second domain may display catalytic activity.
For example, elimination of the critical Cys in D1 of LAR or PTPµ
results in >99% loss of activity (18, 19). The residual activity
(usually less than 0.1%) is concluded to be associated with D2. Recent
evidence suggest that under some conditions D2 of CD45 could be a
viable phosphatase (20), and that certain structural elements in D2 may
be required for full expression of CD45 PTPase activity in D1 (21, 22).
The most compelling data that identify intrinsic phosphatase activity
with D2 come from work on PTP. Working with purified recombinant
individual PTPase domains, Wang and Pallen (23) showed that D2 of
PTP
could hydrolyze p-nitrophenyl phosphate and
dephosphorylate a phosphotyrosine-containing synthetic peptide, albeit
at a very low rate relative to D1. Furthermore, recombinant RPTP
containing an inactive D1 still retained a low but measurable activity
against the Tyr-789-phosphorylated catalytically inactive RPTP
(24).
PTP is a receptor-like PTPase that has a small, highly glycosylated
extracellular segment, a transmembrane region, and an intracellular
segment possessing two PTPase domains (7, 25). PTP
is expressed
ubiquitously, although the levels of expression vary. During mouse
development, enhanced expression was detected in derivatives of the
neural crest (26). Evidence suggest that it may be involved in the
activation of c-src by dephosphorylation of Tyr-527 (16, 27)
and the down-regulation of insulin receptor signaling (28). To better
understand the relative importance of the two PTPase domain in PTP
catalysis, we have compared their intrinsic phosphatase activities
using a variety of low molecular weight aryl phosphates and
Tyr(P)-containing peptides.
p-Nitrophenyl phosphate
(pNPP), -naphthyl phosphate,
O-phospho-L-tyrosine, and 4-methylumbelliferyl
phosphate were purchased from Sigma. 4-Acetylphenyl phosphate,
4-ethoxycarbonylphenyl phosphate, 3-chlorophenyl phosphate, and
4-methylphenyl phosphate were synthesized and characterized as
described earlier (29). Glutathione-agarose beads and human thrombin
were purchased from Sigma. Phosphotyrosine-containing peptides were
synthesized, purified, and characterized as described previously (30).
Peptides DTSSVLpYTAVQ (PDGFR1003-1013),
EGDNDpYIIPL2 (PDGFR1016-1025),
and Ac-DAFSDpYANFK (PTP
784-793) were prepared by the
Laboratory of Macromolecular Analysis of Albert Einstein College of
Medicine. Vanadium (V) oxide (99.99%) was obtained from Aldrich.
Solutions were prepared using deionized and distilled water.
Expression vectors for bacterial
expression of RPTP glutathione S-transferase fusion
proteins were derived by insertion of polymerase chain
reaction-generated NcoI-HindIII fragments into pGEX-KG opened with NcoI and HindIII (31). The
bacterial expression vector encoding the complete cytoplasmic region of
RPTP
(residues 167-793; numbering according to Sap et
al. (25)), bPTP
,3 has been
described (24). The expression vectors for bPTP
-D1 (residues
167-503) and bPTP
-D2 (residues 504-793) were derived using the
oligonucleotide pairs NI, CII and NIV, CI, respectively: NI,
5
-GCGCCATGGCGAAGAAATACAAGCAA, CII,
5
-CCCTCAAGCTTCCAGTTCTGTGTCCCCATA, NIV, 5
-CCCATGGCTTCTCTAGAAACC, CI,
5
-CGCAAGCTTTCACTTGAAGTTGGC. Site-directed mutagenesis was done
on the full-length RPTP
cDNA in pBluscript SK
. Mutations were
verified by sequencing and subsequently the corresponding glutathione
S-transferase fusion protein expression vectors were
constructed as described above. The oligonucleotides that were used for
site-directed mutagenesis are: RPTP
-D401A, 5
-AGCTGGCCAGCCTTTGGGGTG,
and RPTP
-E690D, 5
-GGCTGGCCTGATGTGGGCATC.
The PTP-glutathione
S-transferase fusion protein constructs were transformed
into Escherichia coli BL21(DE3). A 10-ml overnight culture
of BL21(DE3) cells containing pGEX recombinant plasmids for PTP
were
diluted 100-fold into 1 liter of 2×YT medium with 100 µg/ml
ampicillin. The culture was grown at 37 °C until the absorbance at
600 nm reached 0.6. Expression of glutathione S-transferase fusion proteins were then induced by the addition of 100 µM isopropyl-1-thio-
-D-galactopyranoside. Following the induction, the cells were allowed to grow overnight at
room temperature. Purification of the PTP
-glutathione
S-transferase fusion proteins by glutathione-agarose beads
and the subsequent thrombin cleavage of the glutathione
S-transferase fusion protein were accomplished as described
(31). The recombinant PTP
proteins were at least 90% in purity as
judged by SDS electrophoresis.
The PTPase activity was
assayed at 30 °C in a reaction mixture (0.2 ml) containing
appropriate concentrations of pNPP as substrate. Buffers
used were as follow: pH 4.0-5.5, 100 mM acetate; pH
5.5-6.5, 50 mM succinate; pH 6.6-7.3, 50 mM
3,3-dimethylglutarate, and pH 7.5-9.0, 50 mM Tris. All of
the buffer systems contained 1 mM EDTA and the ionic
strength of the solutions were kept at 0.15 M using NaCl.
The reaction was initiated by addition of enzyme and quenched after
2-3 min by addition of 1 ml of 1 N NaOH. The nonenzymatic
hydrolysis of the substrate was corrected by measuring the control
without the addition of enzyme. The amount of product p-nitrophenol was determined from the absorbance at 405 nm
using a molar extinction coefficient of 18,000 M1 cm
1. Michaelis-Menten
kinetic parameters were determined from a direct fit of the v
versus [S] data to the Michaelis-Menten equation (Equation 1)
using the nonlinear regression program GraFit (Erithacus Software).
![]() |
(Eq. 1) |
![]() |
(Eq. 2) |
Initial rates for the PTPase catalyzed
hydrolysis of Tyr(P)-containing peptide substrates were determined by
following the production of inorganic phosphate (30).
kcat and Km values were
calculated from a direct fit of the v versus [S] data to
Equation 1 using the nonlinear regression program GraFit (Erithacus Software). The PTPase-catalyzed hydrolysis of Ty(P)-containing peptides can also be monitored continuously at 305 nm for the increase
in tyrosine fluorescence with excitation at 280 nm (30). When [S]
Km, Equation 1 reduces to Equation 3,
![]() |
(Eq. 3) |
The following procedure for the preparation of stock solution of sodium orthovanadate was adopted from Dr. Mike Gresser's laboratory: vanadium (V) oxide was dissolved in 1 mol eq per vanadium atom of 1.0 M aqueous NaOH. The resulting orange solution (mainly decavanadate) was boiled, allowed to stand overnight, and pH adjusted to 10. The final solution was colorless containing mainly orthovanadate (34). The inhibition constants for inorganic phosphate, arsenate, vanadate, and p-nitrophenol were determined for the phosphatases in the following manner. When oxyanions were used as inhibitors, the initial rate at various pNPP concentrations was measured by following the production of p-nitrophenol (35). When p-nitrophenol was used as an inhibitor, the initial rate at various pNPP concentrations was measured by following the production of inorganic phosphate (33). The inhibition constant and inhibition pattern were evaluated using a direct curve-fitting program KINETASYST (IntelliKinetics, State College, PA).
To study the catalytic
properties of the individual PTPase domains of RPTP, we have
generated glutathione S-transferase fusion protein
constructs that encode the cytoplasmic portions of
RPTP
,4 bPTP
3 (amino acid
residues 167-793), bPTP
-D1 (amino acid residues 167-503), and
bPTP
-D2 (amino acid residues 504-793). A schematic representation
of RPTP
, bPTP
, bPTP
-D1, and bPTP
-D2 is shown in Fig.
1. Glutathione S-transferase fusion proteins
were expressed in E. coli BL21(DE3) and affinity purified on
glutathione-agarose beads. Recombinant bPTP
, bPTP
-D1, and
bPTP
-D2 were cleaved from the agarose bead-bound glutathione
S-transferase with thrombin (31). The purity of bPTP
,
bPTP
-D1, and bPTP
-D2 were general greater than 90% as judged by
SDS-polyacrylamide gel electrophoresis (data not shown). Table
I summarizes the steady-state kinetic parameters of the
various forms of RPTP
using pNPP as a substrate. Thus,
bPTP
(which contains both D1 and D2) exhibits kinetic parameters that are similar to those of bPTP
-D1. Interestingly, the
kcat value for D2 is only 10-fold lower than
that of D1 while the Km is 5-fold higher.
|
We next compared the ability of the individual PTPase domains to
dephosphorylate Tyr(P)-containing peptides corresponding to Tyr
autophosphorylation sites in platelet-derived growth factor receptor
and EGF receptor, or the Tyr phosphorylation site (Tyr-789) in the
COOH-terminal of RPTP (24). The dephosphorylation reaction was
followed by monitoring the production of inorganic phosphate with a
colorimetric procedure (30). Kinetic constants for the hydrolysis of
peptide substrates are listed in Table II. In comparison with the double PTPase domain-containing bPTP
, bPTP
-D1 displays slightly reduced kcat values toward the peptide
substrates, but the Km values are indistinguishable.
However, in contrast to small nonpeptidic substrate pNPP,
the kcat values for the bPTP
-D2-catalyzed hydrolysis of Tyr(P)-containing peptides decrease by 2 to 3 orders of
magnitude compared with bPTP
-D1, while the Km
values increase by 1 to 2 orders of magnitude. Thus, the combined
effects translate into a difference in substrate specificity constant (kcat/Km) in the range of
8,000 to 40,000-fold in favor of bPTP
-D1 over bPTP
-D2.
|
To
begin to gain insight into the structural basis for the differential
activity exhibited by D1 and D2, we have performed an amino acid
sequence alignment for both of the PTPase domains in several
receptor-like PTPases including RPTP (Fig. 2).
Invariant residues in D1 and D2 are listed below the sequence
alignment. It is clear that D1 is more conserved than D2. Previous
biochemical studies have demonstrated that the Cys residue in the
PTPase signature motif
(H/V)C(X)5R(S/T) is essential for
phosphatase activity and formation of a covalent cysteinyl
phosphoenzyme intermediate (39-41). Mutations at the Cys residue
result in complete loss of phosphatase activity. The invariant Arg
residue in the signature motif has been shown to play an important role
in substrate binding and transition state stabilization (5).
Substitutions of Arg-409 in the Yersinia PTPase by either a
Lys or an Ala cause a decrease in kcat by nearly
4 orders of magnitude. In addition to nucleophilic catalysis and
transition state stabilization, PTPases also utilize an invariant Asp
residue in the WPD motif (Fig. 2) to facilitate the catalytic turnover:
the Asp residue acts as a general acid to donate a proton to the
phenolate leaving group in the phosphoenzyme formation step, while it
acts as a general base to activate a nucleophilic water molecule in the
phosphoenzyme hydrolysis step (4, 42). Replacement of Asp-356 by an Asn
in Yersinia PTPase reduces the turnover number by 3 orders
of magnitude. As can be seen in Fig. 2, the PTPase signature motif and
the WPD motif are strictly conserved among the D1 domains. These two
motifs are not conserved among the D2 domains.
A closer examination of the sequence alignment shows that in the D2
domain of PTP, the essential Cys and Arg residues in the active site
are replaced by an Asp and a Ser, respectively. Furthermore, the Asp
residue in the WPD motif is replaced by an Asn. Thus, it is unlikely
that the second PTPase domain of PTP
will possess any catalytic
activity. Although the active site Cys is retained in the D2 domain of
CD45, it lacks the critical Arg residue while the Asp residue is
substituted by a Val. It is thus questionable whether the second PTPase
domain of CD45 will exhibit any significant phosphatase
activity.5 Interestingly, the D2 domains of
PTP
, PTP
, and LAR preserve all of the essential features of the
PTPase signature motif, but the Asp residue in the WPD motif has been
switched to a Glu. Based on the sequence alignment, Asp-401 is the
putative general acid/base in the WPD motif in bPTP
-D1 whereas
Glu-690 is the putative general acid/base in the WPE motif in
bPTP
-D2. It is found that replacement of Asp with Glu or vice versa
generally leads to a reduction of up to 3 orders of magnitude in
catalytic efficiency in enzymes that require a carboxylate group as a
general acid/base (43-46). Since an Asp is always found to be the
general acid/base in the catalytic domains of cytoplasmic PTPases and
the D1 domains of receptor-like PTPases, and since the D2
domains of receptor-like PTPases have much reduced
catalytic activity, we asked the following question: can substitution
of Glu-690 by an Asp in bPTP
-D2 increase its catalytic activity to a
level comparable to bPTP
-D1?
To answer this question, we have converted Glu-690 in bPTP-D2 to an
Asp by site-directed mutagenesis. We have also made the Asp-401 to Ala
mutation in bPTP
-D1 to test the importance of the Asp residue in the
WPD motif. Although similar site-directed mutagenesis experiments with
the invariant Asp residue have been performed with intracellular
PTPases (4, 47), such experiments have not been done with
receptor-like PTPases. Both bPTP
-D1/D401A and bPTP
-D2/E690D were
expressed and purified similarly to near homogeneity as described for
the wild-type D1 and D2. Using pNPP as a substrate, the
kcat for bPTP
-D1/D401A is 900-fold slower than that of wild-type bPTP
-D1, while the Km for
bPTP
-D1/D401A is identical to that of bPTP
-D1 (Table I). These
results are similar to those observed with cytoplasmic PTPases and
suggest that Asp-401 is indeed important for the activity of D1 and
that a common catalytic strategy is shared by both the receptor-like and soluble PTPases.
Asp substitution for Glu at 690 in bPTP-D2 has modest effects on the
kinetic parameters using pNPP as a substrate (Table I). A
4-fold increase in kcat and nearly identical
Km were observed for bPTP
-D2/E690D when compared
with the wild-type D2. In fact, bPTP
-D2/E690D has a
kcat value that is only 2-fold lower than that
of bPTP
-D1 using pNPP as a substrate. Similar results
were obtained with the Tyr(P)-containing peptides (Table II), namely
D2/E690D displays 3-4-fold higher kcat value
and identical Km value in comparison with the native
D2 domain. However, the kcat and
kcat/Km values of
bPTP
-D2/E690D toward Tyr(P)-containing peptides are still 3 and 4 orders of magnitude lower when compared with bPTP
-D1 (Table II).
These results suggest that substitution of Glu-690 for an Asp in D2 may
not be the main cause that D2 is a much less efficient phosphatase than
D1 toward phosphopeptide substrates.
Inhibition constants were determined for inorganic
phosphate, arsenate, vanadate, and p-nitrophenol at pH 6.0 (Table III). In general, effects of inhibitors to
bPTP-D1-catalyzed reaction are similar to that catalyzed by the
double domain construct bPTP
. Substitution of Glu-690 by an Asp does
not enhance significantly the affinity of bPTP
-D2 toward the
oxyanions. Oxyanions were competitive inhibitors with respect to
pNPP, whereas p-nitrophenol was noncompetitive.
The pattern of product (phosphate and p-nitrophenol) inhibition suggests that D1 and D2 utilize a similar kinetic scheme (Scheme 1), particularly in terms of the order of
addition of substrates and release of products (33). Inorganic
phosphate usually shows Ki values around 1-5
mM against PTPases (33, 42, 48). Compared with other
PTPases, bPTP
-D1 binds phosphate with an unusually weak affinity
(Ki = 120 mM) which is even 2-fold lower
than bPTP
-D2. Arsenate binds to bPTP
-D1 and bPTP
-D2 more
tightly than phosphate, however, the increase in affinity is seen
greater with bPTP
-D1 than bPTP
-D2. Thus, arsenate inhibits the
bPTP
-D1-catalyzed reaction (Ki = 0.25 mM) 30-fold more potently than the bPTP
-D2-catalyzed
reaction.
|
Vanadate is by far the most potent small molecule inhibitor for
PTPases. Because vanadate can adopt five-coordinate structures readily,
it was expected that a covalent bond can be formed between the active
site thiol and the vanadium atom. The structure of Yersinia
PTPase complexed with vanadate revealed continuous electron density
between the bound anion and the active site Cys-403, consistent with a
covalent bond (49). Indeed, vanadate in the active site adopts a
slightly distorted trigonal bipyramidal geometry that resembles the
transition state for the hydrolysis of the thiophosphate enzyme
intermediate in the PTPase-catalyzed reaction (6, 50). Previous studies
with vanadate have often included EDTA in the assay buffers. Since EDTA
forms a very stable 1:1 complex with vanadate even at micromolar
concentrations of both EDTA and vanadate (51), the presence of EDTA in
the assay solution will reduce the free vanadate concentration. Here we
performed all of our inhibition experiments with vanadate in the
absence of EDTA. Vanadate shows an apparent Ki of
5.5 µM toward bPTP-D1 whereas the
Ki for bPTP
-D2 is 1.9 mM. Thus,
bPTP
-D1 binds vanadate 350-fold tighter than bPTP
-D2.
Furthermore, bPTP
-D1 binds vanadate (a crude transition state
analog) 22,000-fold more tightly than phosphate (a crude substrate
analog), while bPTP
-D2 binds vanadate only 40-fold more tightly than
phosphate. Since one of the major means an enzyme utilizes to catalyze
a reaction is via transition state stabilization, it is understandable
that bPTP
-D1 is a better PTPase than bPTP
-D2. Because vanadate
can take on different forms in solution, and because the enzyme-bound
vanadate has a different structure from the tetrahedral vanadate in
solution, it is very likely that the kinetically determined
Ki may underestimate how good a transition state
analog the PTPase-bound vanadate really is (52).
The pH dependence of the steady-state kinetic
parameters for the various forms of PTP were studied in detail using
pNPP as a substrate. Within the pH range investigated,
Km values only exhibited moderate variations from
those reported in Table I. The pH-kcat profile
is shown in Fig. 3. The line that is drawn through the
experimental data is based on the nonlinear least-squares fit of the
data to Equation 2. pK1app,
pK2app, and the
pH-independent maximum turnover number
kcatlim are listed in Table
IV. It appears that bPTP
-D1 displays a similar pH
rate profile to that of bPTP
, except
kcatlim for bPTP
is twice
that of bPTP
-D1. Although substitution of an Asp in place of Glu-690
in bPTP
-D2 increase the
kcatlim by 4-fold, it does
not alter the pH rate profile. It is also clear that bPTP
-D1 and
bPTP
-D2 display different pH rate profile: the pH optimum for
bPTP
-D1-catalyzed hydrolysis of pNPP is 0.5 pH unit lower
than that of bPTP
-D2.
|
We have shown above that when
compared with bPTP-D1, the kcat and
kcat/Km values of bPTP
-D2
toward pNPP are reduced by 10- and 50-fold, respectively
(Table I). On the other hand, when Tyr(P)-containing peptides are used
as substrates, up to 3 and 4 orders of magnitude reduction are observed
with kcat and kcat/Km, respectively (Table
II). One possible explanation for this differential effects may be that
Glu-690 in bPTP
-D2 is a weaker general acid than an Asp in
bPTP
-D1, so that substrates with Tyr (pKa = 10.07) as a leaving group becomes more difficult to expel because the
negative charge developed on the phenolate oxygen in the transition
state (47) cannot be efficiently stabilized by Glu-690. Leaving groups
in substrates such as pNPP (pKa = 7.14)
are much easier to expel since there is much less demand for general
acid catalysis.
To test this hypothesis, we investigated the leaving group dependence
of the PTPase-catalyzed hydrolysis of aryl phosphates. Since
Tyr(P)-containing peptides are more complex and different from
pNPP in terms of structure, we chose several low molecular weight aryl phosphates to minimize the influence on the reaction by
steric effects. As shown in Fig. 4, both bPTP and
bPTP
-D1 exhibit no leaving group dependence and show very similar
kcat values for hydrolysis of aryl phosphates
that differ markedly in their leaving group pKa (for
example, 7.14 for p-nitrophenol and 10.26 for
4-methylphenol). Interestingly, bPTP
-D2 and bPTP
-D2/E690D display
only moderate leaving group dependences, with
lg values of
0.092 ± 0.041 and
0.11 ± 0.051, respectively. Thus
although bPTP
-D2/E690D is four times more active than bPTP
-D2, it
does not alter the leaving group sensitivity. From these observations, it appears that the 3-4 orders of magnitude decrease in reactivity toward Tyr(P)-containing peptides by bPTP
-D2 and bPTP
-D2/E690D is
not due to the intrinsic lower stability of the Tyr leaving group. This
is because, in comparison with pNPP, one would only predict
a 2-fold decrease in rate for phosphotyrosine based on the leaving
group dependences of bPTP
-D2 and bPTP
-D2/E690D catalyzed aryl
phosphate hydrolysis reaction.
Peptide Substrate Amino Acid Sequence Specificity
Substrate
specificity in terms of peptide size and amino acid sequence preference
were investigated using Tyr(P)-containing peptides. The
PTPase-catalyzed dephosphorylation reaction was continuously monitored
spectrofluorimetrically (excitation at 280 nm and emission at 305 nm)
at pH 6.0 and 30 °C. This assay takes advantage of the increase in
fluorescence at 305 nm when the phosphate is hydrolyzed from Tyr(P)
(30). At substrate concentrations Km, the
Michaelis-Menten equation (Equation 1) reduces to Equation 3, and the
reaction is first order with respect to [S]. With automatic data
collection and nonlinear least square fit analysis, the first-order
rate constant can be determined with a precision of less than 4%. The
observed apparent first-order rate constant is equal to
(kcat/Km)[E],
from which the substrate specificity constant
kcat/Km value can be
calculated by dividing the apparent first-order rate constant with the
enzyme concentration. Additional advantages of this assay include that very small amount of peptide substrate is used per reaction and that
accurate substrate concentration determination is not necessary. In
general, substrate concentrations were at least 15-fold lower than the
Km values (Table II) and ranged from 2 to 10 µM. To ensure first-order kinetics, reactions were
conducted at several substrate concentrations. Since the reaction is
first order in nature, the same rate constant is obtained regardless the substrate concentration used. Because the
kcat/Km values for bPTP
-D2
and bPTP
-D2/E690D are 5 orders of magnitude slower than those of
bPTP
, they cannot be determined by the fluorescence assay.
The peptide substrate amino acid sequence specificity was probed with
Tyr(P)-containing peptides corresponding to Tyr autophosphorylation sites in platelet-derived growth factor receptor, EGF receptor, Neu
oncogene, Src kinase, or RPTP (Table V). The
substrate specificity constants
(kcat/Km values) determined
by the fluorescence assay are very similar to those calculated from the
ratio of kcat and Km, which
are determined by the initial rate method (Table II). In agreement with
results described above, bPTP
and bPTP
-D1 display similar
substrate specificity toward peptide substrates, but the
kcat/Km values for bPTP
are slightly higher that those of bPTP
-D1, which is likely due to
the slightly higher kcat for bPTP
. In
general, bPTP
and bPTP
-D1 only exhibit a moderate selectivity
toward the various peptides. Only a 8-fold difference in
kcat/Km was observed among
the peptides. Although the enzyme does appear to prefer peptides with
two acidic residues immediately NH2-terminal to Tyr(P)
(DADEpYLIPQQG and DAEEpYLVPQQG), there is also exception to this
(DTSSVLpYTAVQ).
|
To determine the
minimum length of a peptide substrate for bPTP and bPTP
-D1, we
selected a series of various sized phosphopeptides modeled after the
autophosphorylation site in EGF receptor (EGFR) at Tyr-992 (Table
VI). For the sake of discussion, we will focus the
results on bPTP
, since identical conclusions can be reached from
results on bPTP
-D1. Phosphotyrosine (Tyr(P)) by itself is not a good
substrate. In fact, its
kcat/Km value cannot be
determined by the fluorescence assay because of the slow rate. We
therefore determined its kcat and
Km by the initial rate method using a molar
absorption coefficient of 2,400 for tyrosine (29). The calculated
kcat/Km values for Tyr(P) are
listed in Table VI. It seems that the low
kcat/Km for Tyr(P) is
primarily due to its high Km: 22.1 mM
for bPTP
(19.5 mM for bPTP
-D1). As shown in Table VI,
the kcat/Km value for the
parent peptide DADEpYLIPQQG (EGFR988-998) is 1090-fold
higher than Tyr(P). This indicates that structural elements surrounding
Tyr(P) in the peptide contribute to specificity.
|
Strikingly, Ac-Tyr(P)-NH2, which is obtained by acetylating the free amino group and amidating the free carboxyl group of Tyr(P), exhibit a kcat/Km that is 76-fold higher than Tyr(P). Interestingly, the tripeptide EpYL is only 4.7-fold better than Tyr(P). Since acetyl group is smaller than a Glu residue, the dramatic enhancement in substrate efficacy for Ac-Tyr(P)-NH2 seems to mainly arise from the elimination of charges on the free amino and carboxyl groups, rather than the increase in size. The addition of an Ile to the COOH-terminal side of EpYL (generating EpYLI) results in only a 1.9-fold improvement, while addition of an Asp to NH2-terminal side of EpYL (generating DEpYL) leads to a 1.5-fold increase in activity. Although the acetyl group is smaller than an Asp residue, the kinetic parameter for Ac-EpYL is 17-fold higher than that of DEpYL. This may again be due to the elimination of the positive charge on the NH2-terminal amino group of EpYL.
The incorporation of an additional residue, Ile, to the COOH-terminal
side of DEpYL produces a pentapeptide (DEpYLI) that is 56 times more
efficient than DEpYL. Thus, the extension of DEpYL beyond the +1
position by one residue is more than enough to offset the undesirable
charge effect on the NH2-terminal side. Further extension
at the NH2-terminal of DEpYLI by an Ala (to give ADEpYLI)
improves its efficacy by 1.8-fold. Attachment of a Pro residue to the
COOH-terminal of peptide ADEpYLI does not produce significant increase
in substrate specificity, whereas attachment of an acetyl group to the
NH2-terminal of peptide ADEpYLI actually reduces its
efficiency slightly (by 1.3-fold). This suggests that the hexapeptide
ADEpYLI may represent a minimal peptide substrate for bPTP and
bPTP
-D1. In fact, the
kcat/Km value for ADEpYLI is
within 1.5-fold of the parent peptide DADEpYLIPQQG. We are surprised by
the observation that in the absence of additional residues beyond the
+2 position in ADEpYLI, elongation at the NH2-terminal side
(e.g. Ac-DADEpYLI) interferes with bPTP
activity. However
Ac-DADEpYLI is still about twice as efficient as Ac-DADEpYL, consistent
with the importance of residues at the +2 position noted above.
We also investigated whether residues on the NH2- or
COOH-terminal side of Tyr(P) are more important in determining bPTP substrate specificity. When all of the residues
NH2-terminal to the Tyr(P) of EGFR988-998 are
removed, the resulting peptide pYLIPQQG is such a poor substrate that
its kcat/Km value cannot be
determined by the fluorescence method. Due to the limited amount of
material, we could not measure its kcat and
Km by the initial rate method. Since the lowest
kcat/Km value determined by
the fluorescence assay is 690 M
1
s
1 (for the bPTP
-D1-catalyzed hydrolysis of EpYL), we
assume it as an upper limit for pYLIPQQG. Thus, pYLIPQQG behaves
kinetically similarly to phosphotyrosine. This could either indicate
that positive charge on the amino group of Tyr(P) is deleterious or that residues COOH-terminal to Tyr(P) are not important for binding. Judging from the results described above, the former possibility is
most likely. Further experiments are required to resolve this. Interestingly, when all of the residues COOH-terminal to the Tyr(P) are
removed from EGFR988-998 and the negative charge on the
free carboxylate eliminated by amidation, the
kcat/Km value of the
pentapeptide DADEpY-NH2 approaches that of the parent
peptide. Surprisingly, the hexapeptide DADEpYL-NH2, which
is obtained by incorporating just one more Leu residue to the
COOH-terminal side of Tyr(P) in DADEpYL-NH2, shows a 4-fold
decrease in substrate specificity in comparison with the pentapeptide.
Thus, it appears that in the absence of charge and additional residues
on the COOH-terminal side of Tyr(P), four residues
NH2-terminal to Tyr(P) can serve as an effective substrate.
Collectively, from a systematic analysis of different fragments of
EGFR988-998 peptide, we conclude that efficient binding
and catalysis by bPTP
and bPTP
-D1 require minimally either six
amino acids (including Tyr(P), three residues NH2- and two
residues COOH-terminal to Tyr(P)) or five amino acids (including
Tyr(P)-NH2 and four residues NH2-terminal to
Tyr(P)).
PTP is a representative of the receptor-like PTPases that
contains two PTPase domains. In this paper we try to address whether both of the PTPase domains in PTP
are active, and if both are active, what are the relative contributions for each domain to the
double domain molecule. We have compared the kinetic properties of
bPTP
, bPTP
-D1, and bPTP
-D2 using near homogeneous preparations of recombinant proteins. We demonstrate that bPTP
-D1 and bPTP
(containing two tandem PTPase domains) display similar steady-state kinetic parameters, pH and leaving group dependences, sensitivity to
oxyanion and p-nitrophenol inhibitors, and substrate
specificity. This suggests that the catalytic activity of PTP
is
dominated by the D1 domain and that the D2 domain does not contribute
significantly to the overall property of the double domain construct.
This is likely due to the fact that the kcat for
D2 is much reduced and the Km for D2 is much
elevated when compared with those of D1, especially in cases of
phosphopeptide substrates.
The three-dimensional structure of RPTP-D1 (containing residues
202-503) has been determined (53). This particular D1 construct crystallized as a homodimer. It was found that a segment of the NH2-terminal sequence (residues 208-242) of one monomer
formed a helix-turn-helix structural wedge which tucked into the active site of the dyad-related monomer. This restrained the WPD loop of each
monomer in the open (inactive) conformation and would block potential
substrate binding. The D1 construct used in this study contains amino
acids from 167 to 503 (Fig. 1). We find that under our assay conditions
both bPTP
-D1 and bPTP
are active. Furthermore, at bPTP
-D1
concentration between 10 and 100 nM, the apparent
first-order rate constant for the hydrolysis of Tyr(P)-containing peptides is strictly first-order with respect to the enzyme
concentration with a zero y intercept when the rate constant
is plotted against enzyme concentration (data not shown), consistent
with bPTP
-D1 exists as an active monomer in solution.
We also demonstrate that the D2 domain of PTP is a genuine PTPase in
its own right. This is especially true when low molecular weight aryl
phosphates are used as substrates. In fact, the
kcat value for D2 is only 10-fold lower than
that of D1 while the Km is 5-fold elevated with
pNPP as a substrate. However, the D2-catalyzed reaction
shows different pH and leaving group dependences from that of D1. It
also possesses a much lower affinity to the transition state analog
vanadate and greatly reduced kcat and
kcat/Km values for peptide
substrates. In a previous study, the Vmax and Km values for the D2 domain of human
PTP
-catalyzed hydrolysis of a Tyr(P)-containing peptide (RR-Src)
were shown to be 200-fold lower and 47-fold higher, respectively, when
compared with the D1 domain (23). These differences in
kcat and Km between D1 and D2
fall within the range that we observed with peptide substrates.
However, from the same study, the Vmax value for
the D2-catalyzed hydrolysis of pNPP was shown to be 1.6-fold
higher than that of D1. We do not know what causes the discrepancy
regarding the pNPP data, except to note that in the previous
study, D1 domain included residues from 167 to 542 whereas D2 domain
included residues from 487 to 774 (23), which are different from the
constructs we made in this work (Fig. 1).
We hypothesized that the large differences in catalytic properties
between D1 and D2 might be caused by structural variations between the
active sites of the two PTPase domains. Amino acid sequence alignment
of both D1 and D2 from a number of receptor-like PTPases reveals that
the D2 domain of PTP contains all the essential features found in
the PTPase signature motifs of cytoplasmic PTPases and the D1 domains
of receptor PTPases. In addition to the fact that D1 domains are more
conserved and homologous to the catalytic domains of cytoplasmic
PTPases than D2 domains, the most apparent difference is that the
putative general acid (Asp residue) in the WPD motif observed in all D1
domains and all cytoplasmic PTPases has been changed to Glu-690 in
bPTP
-D2. We illustrate that mutation of Asp-401 in the WPD motif of
bPTP
-D1 to an Ala decreases the kcat by
900-fold, confirming the importance of general acid/base in the D1
domain of receptor PTPases. Replacement of Asp with Glu or vice versa
can have dramatic effects in enzymes that require a carboxylate group
as a general acid/base (43-46). Interestingly, the restoration of the
general acid/base to an Asp at position 690 in bPTP
-D2 only raises
the activity by 4-fold. This is consistent with our previous mutational
studies of the general acid Asp-128 in the low molecular weight PTPase:
D128E retained 15% of the native catalytic activity (42). Furthermore,
substitution of Glu-690 by an Asp in bPTP
-D2 does not alter its pH
and leaving group dependences, its sensitivity to oxyanion inhibitors,
and its substrate specificity. Collectively, these results indicate that the active site of PTPase is fairly flexible and can tolerate a 1 methylene unit lengthened side chain as long as the carboxylate functionality is preserved.
It is interestingly to note that the phosphatase activity of D2 toward
aryl phosphates are comparable to that of D1, but its activity toward
Tyr(P)-containing peptides/proteins is much reduced (this work and
Refs. 23 and 24). Since the kcat for bPTP-D2 catalyzed hydrolysis of 4-methylphenyl phosphate
(pKa of 4-methylphenol = 10.26) is only
2-3-fold slower than that of pNPP (pKa
of p-nitrophenol = 7.14), the very low activity toward
peptide substrates is unlikely due to the higher pKa of tyrosine (10.07). This together with the fact that Glu-690 to Asp
substitution did not restore D2's activity to D1 suggest structural
features other than the general acid/base residue may be responsible
for the low activity toward peptide substrates. A three-dimensional
structure for the D2 domain may shed more light on this. On the other
hand, the reduced activity toward peptide substrates may also suggest
that bPTP
-D2 has a functional active site with a highly stringent
substrate selectivity. Thus, the configuration of active site residues
in D2 responsible for binding and/or catalysis may not be optimal for
nonspecific peptide substrates that require extensive intermolecular
interactions. The possibility that there exists physiological
substrate(s) for the D2 domain of PTP
remains to be explored.
The kcat values for the bPTP and bPTP
-D1
catalyzed hydrolysis of aryl phosphates and Tyr(P)-containing peptides
are similar. This is consistent with the rate-determining step being
the hydrolysis of the phosphoenzyme intermediate (E-P in
Scheme 1) (6). The rate-limiting step for the D2-catalyzed hydrolysis
of low molecular weight aryl phosphates may be predominately determined
by E-P hydrolysis as well, given the small
lg
value observed. The fact that kcat values for
phosphopeptides are 40-160-fold slower than small aryl phosphates such
as pNPP in the D2-catalyzed reactions suggest that the
rate-limiting step for peptide substrates may be changed to the
formation of E-P. This is consistent with the notion that
the D2 domain may recognize a global conformation and require a precise
alignment between residues in the enzyme active site and the specific
substrate. Nonspecific peptide substrates may not be capable of forming
productive enzyme-substrate complexes for the forward reaction.
Detailed kinetic analysis of bPTP and bPTP
-D1 catalyzed
Tyr(P)-containing peptides show moderate selectivity toward different substrates. Our data also indicate that positive charges from the free
amino group at Tyr(P),
1, and
2 positions and the negative charge
of the free carboxylate at Tyr(P) can be detrimental to PTP
catalysis. We have shown previously that in the sequence context of
DADEpYLIPQQG (EGFR988-998), the Yersinia
PTPase, and the mammalian PTP1 require a minimum of six amino acid
residues for most efficient binding and catalysis (54). These include
Tyr(P), four amino acid residues NH2-terminal, and one
amino acid residue COOH-terminal to the Tyr(P)
(DADEpYL-NH2). Using the same series of peptides, we
conclude that for optimal PTP
activity the minimal size of peptide
substrates can either be ADEpYLI or DADEpY-NH2. Thus,
different PTPases may possess different active site specificity in
terms of short peptide substrates. It appears that short optimal
peptides can be accommodated in the active site of the D1 domain by
more than one mode. Furthermore, the recognition between PTP
and
short peptide substrates seems sensitive to structural
modifications.
Further understanding of the specific functional roles of PTPases in cellular signaling requires detailed investigation of PTPase substrate specificity. Clearly, a more systematic and thorough approach, such as the use of degenerate peptide libraries containing randomized amino acid residues surrounding the phosphotyrosine, is needed for the search of consensus sequence motif for individual PTPases. Although consensus peptide motifs of many protein kinases have been elucidated (55), those of PTPases are essentially unknown. Specific consensus peptide motif for PTPases will lay the foundation for structure based rational PTPase inhibitor design. Since the identity of the physiological substrates for most PTPases is not known at present, such optimal substrate motifs may also be useful for the identification of physiological substrates for PTPases from available protein sequence data bases.
Finally, based on the kinetic data and the comparison of the amino acid
sequence alignment of D1 and D2 from a number of receptor-like PTPases
presented in this paper, we suggest that D2 domains of certain types of
receptor-like PTPases (e.g. PTP, PTP
, and LAR) should
display significant intrinsic phosphatase activity whereas D2 domains
of other PTPases may possess little (e.g. CD45) or no
(e.g. PTP
) phosphatase activity. The functional role of
the second domain, i.e. whether it regulates the specificity
and activity of the first domain and/or exhibits independent substrate
selectivity, requires further investigations.