(Received for publication, August 7, 1995; and in revised form, November 17, 1995)
From the
Photoaffinity labeling has been used to identify amino acids
involved in recognition of protein substrates by the protein-tyrosine
phosphatase PTP1. The photoactive amino acid p-benzoylphenylalanine (Bpa) was incorporated into a
phosphotyrosine-containing peptide derived from epidermal growth factor
autophosphorylation site Tyr (EGFR
). This peptide photoinactivated
PTP1 in a time- and concentration-dependent manner. Three lines of
evidence indicate that the interaction between PTP1 and the
photoaffinity label was specific: 1) photoinactivation was inhibited in
the presence of a non-Bpa-containing peptide from EGFR Tyr
in molar excess. 2) The photoaffinity label-containing
phosphopeptide was rapidly dephosphorylated by PTP1 with kinetic
constants similar to those of the non-Bpa-containing peptide under
identical conditions. 3) After complete photoinactivation, the level of
incorporation of radioactive photoaffinity label into PTP1 was
approximately 0.9 mol of label/mol of enzyme, consistent with a 1:1
stoichiometry of photolabeling. Radiolabeled peptide was used to
identify sites of cross-linking to PTP1. Bpa peptide-PTP1 was digested
with trypsin, and radioactive fragments were purified by high
performance liquid chromatography (HPLC) and analyzed by Edman
sequencing. In two parallel experiments which were analyzed using
different HPLC columns, a site in the
2` region of PTP1, most
likely Ile
, was labeled by the Tyr
-derived
peptide. The results are discussed in light of the crystal structure of
human PTP1B and suggest that an additional mode of substrate
recognition must exist for PTP1 catalysis.
Tyrosine phosphorylation of proteins is a fundamental mechanism
for the control of cell growth and differentiation. In vivo,
this process is reversible and dynamic; the phosphorylation states of
proteins are governed by the opposing actions of protein-tyrosine
kinases, which catalyze protein-tyrosine phosphorylation, and
protein-tyrosine phosphatases (PTPases), ()which are
responsible for dephosphorylation(1, 2) . The
functional role of PTPases in cellular signaling processes is just
beginning to be appreciated(3) . PTPases constitute a growing
family of transmembrane (receptor-like) and intracellular enzymes that
rival the protein tyrosine kinases in terms of structural diversity and
complexity. 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) .
A central question in the field is how PTPases distinguish the diversity of substrates that they encounter in the cell. Despite the rapid progress in the identification and characterization of new PTPases, there have been relatively few biochemical analyses of the mechanisms that govern PTPase substrate specificity. Evidence suggests that both catalytic and noncatalytic regions of PTPases are important for phosphotyrosyl substrate recognition. In some cases, noncatalytic domains localize the enzymes to specific intracellular compartments in which the effective local concentration of a substrate is high. For example, a COOH-terminal extension of the PTP1B tyrosine phosphatase has been shown to be necessary and sufficient for targeting the enzyme to the cytoplasmic side of the endoplasmic reticulum(5) . In other cases, noncatalytic segments of PTPases play a role in modulating enzyme activity in an allosteric fashion. Occupancy of both SH2 domains of the PTPase SH-PTP2 (also known as Syp, PTP1D, or PTP-2C) stimulates phosphatase activity(6, 7) . Thus, cellular SH-PTP2 activity and specificity is regulated by the interaction of the enzyme with phosphotyrosine-containing proteins that bind to its SH2 domains.
PTPase substrate specificity must also be mediated by intrinsic
substrate specificities of the active site as well as structural
features in the vicinity of the phosphorylated tyrosine residue. Using
synthetic phosphotyrosine-containing peptides that correspond to
natural phosphorylation sites in proteins, several groups have
demonstrated that PTPases display a range of k/K
values for
these relative short peptide
substrates(8, 9, 10, 11, 12) .
In fact, the k
/K
values for some of the peptide substrates approach the
efficiency limited by diffusion events, suggesting that short optimal
phosphopeptide sequences may contain all the information that is needed
for in vivo PTPase recognition(9, 10) .
The human PTP1B is the founding member of the PTPase
family(13) . The three-dimensional structure of the catalytic
domain (residues 1-322) of PTP1B has been
determined(14) . The structural homolog of the human PTP1B from
rat, PTP1, is one of the most extensively studied PTPases (9, 10, 15, 16, 17, 18) .
The catalytic domain of PTP1 (residues 1-322) is 97% identical to
the corresponding 322 residues of the human PTP1B. We have shown
previously that amino acid residues flanking the phosphotyrosine are
important for efficient PTP1 catalysis ( Table 1and Refs. 9, 10,
and 17). For example, the k/K
value for the undecapeptide, EGFR
(epidermal growth factor autophosphorylation site
Tyr
, residues 988-998)
(Asp-Ala-Asp-Glu-pTyr-Leu-Ile-Pro-Gln-Gln-Gly) is 3220-fold higher than
that of phosphotyrosine (Table 1). We further demonstrated that a
minimum of six amino acid residues are required for the most efficient
PTP1 binding and catalysis. These include phosphotyrosine, four amino
acid residues NH
-terminal and one amino acid residue
COOH-terminal to the phosphotyrosine. Indeed, the hexapeptide,
Asp-Ala-Asp-Glu-pTyr-Leu-NH
, is an excellent PTP1 substrate
that exhibits a k
/K
of 2.24
10
M
s
(17) . The recently solved crystal
structure of the active site Cys
to Ser mutant of PTP1B
complexed with the high-affinity substrate
Asp-Ala-Asp-Glu-pTyr-Leu-NH
(19) reveals specific
interactions between acidic side chains of the substrate and basic
residues of the enzyme. However, kinetic studies have demonstrated that
PTP1 can hydrolyze a variety of peptide substrates differing in
sequence and length with almost equal k
/K
values(9, 10, 17) . For example,
the peptide substrates Neu
(Asp-Asn-Leu-Tyr-pTyr-Trp-Asp-Gln-Asn-Ser-Ser) and
p60src
(Thr-Glu-Pro-Gln-pTyr-Gln-Pro-Gly-Glu)
display kinetic parameters similar to those of EGFR
(Asp-Ala-Asp-Glu-pTyr-Leu-Ile-Pro-Gln-Gln-Gly) (Table 1).
These results cannot be easily explained by the observed interactions
in the crystal structure of PTP1B complexed with
Asp-Ala-Asp-Glu-pTyr-Leu-NH
and suggest that additional
determinants for peptide substrate recognition by PTP1 must exist.
In order to investigate further the molecular mechanism of substrate
recognition, we have in the present study carried out photoaffinity
labeling experiments on the native form of PTP1 to identify region(s)
of the enzyme involved in peptide substrate binding. The photoactive
probe used was a phosphotyrosyl peptide derived from epidermal growth
factor (EGFR) autophosphorylation site
Tyr
in which the Glu residue immediately
NH
-terminal to the phosphotyrosine was replaced with the
amino acid p-benzoylphenylalanine
(Bpa)(20, 21) . This photoactive amino acid, like
other benzophenone-type labels, will cross-link efficiently to a wide
range of binding sites in proteins (22, 23, 24) . Our results lead to the
identification of an additional region of PTP1 which is involved in
substrate recognition.
To obtain kinetic parameters for the PTP1-catalyzed hydrolysis of the phosphorylated peptide 992N-Bpa, a continuous spectrophotometric assay described previously (9) was employed. This assay takes advantage of the difference in absorbance at 282 nm between phosphotyrosine and tyrosine and can be utilized to follow the complete time course of the enzyme catalyzed hydrolysis of phosphotyrosine containing peptide. The complete time course of the reaction can be fitted to the integrated form of the Michaelis-Menten equation () using a nonlinear least squares algorithm. All of the experiments were performed at 30 °C in either pH 6.6 or pH 8.0 buffers. Buffers used were as follows: pH 6.6, 50 mM 3,3-dimethylglutarate, and pH 8.0, 50 mM Tris. Both buffer systems contained 1 mM EDTA and the ionic strength of the solutions were kept at 0.15 M using NaCl.
Radioactive peaks from the modified digests were concentrated in a Speedvac concentrator (Savant) and analyzed by Edman peptide sequencing using an Applied Biosystems model 475 A pulsed liquid protein sequencer. The samples were concentrated onto an Ultrafree device containing Immobilon-CD membranes (Millipore) by centrifugation at 800 rpm for 15 min. Precycling prior to normal sequencing cycles involved the following successive treatments: trifluoroacetic acid, dry, N-heptane, butyl chloride, dry, trimethylamine buffer, dry, phenylisothiocyanate in N-heptane, trimethylamine buffer, dry, N-heptane, ethyl acetate. For each peak sequenced, portions of the filtrate from the Ultrafree device, of the washes and of the precycling samples, were analyzed by scintillation counting. These samples contained negligible radioactivity (cpm-blank = 10). At each sequencing cycle, one-half of the phenylthiohydantoin-derivative was identified by HPLC and the remainder was dissolved in ScintiVerse fluid and analyzed by scintillation counting.
Figure 1:
A,
photoinactivation of PTP1 by Peptide 992N-Bpa. Enzyme (5
µM) and photoaffinity label (at 0, 100, or 500
µM) were combined in 50 mM Tris (pH 8.0) and
irradiated at 350 nm for up to 15 min. Aliquots were withdrawn and
analyzed for tyrosine phosphatase activity toward pNPP as
described in the text. 100% activity was defined as the activity of the
enzyme/peptide mixture without photolysis; the actual value of 100%
activity was 4.8 10
nmol/min/mg enzyme.
, 0
µM;
, 100 µM;
, 500
µM. B, protection from photoinactivation by
Peptide EGFR
. Photoinactivation was carried out
as above with 5 µM PTP1 and 50 µM Peptide
992N-Bpa, in the presence or absence of an excess (1.5 mM) of
Peptide EGFR
. Enzyme activity was analyzed as
described in the text.
, +EGFR
;
, no EGFR
.
Further evidence supporting this binding of the Bpa-containing peptide to the active site was obtained by testing the peptide as a substrate. PTP1 (238 nM) was incubated with 1.8 mM Peptide 992N-Bpa under the activity assay conditions described above. Aliquots were withdrawn after 1 and 10 min of reaction and analyzed by high performance liquid chromatography. The phosphorylated peptide (Peptide 992N-Bpa) eluted at 26.9 min under these HPLC conditions (Fig. 2). The elution time of the dephosphorylated peptide 992N-Bpa was determined to be 28.1 min by incubating the peptide with potato acid phosphatase in 50 mM sodium citrate (pH 5.0) and analyzing on the HPLC (data not shown). After 10 min of reaction, the starting phosphotyrosyl peptide was converted to material with the HPLC mobility of authentic unphosphorylated peptide (Fig. 2). Thus, PTP1 completely dephosphorylates the peptide under these assay conditions, arguing that Peptide 992N-Bpa binds to the enzyme's active site.
Figure 2: Dephosphorylation of Peptide 992N-Bpa. Peptide 992N-Bpa (1.8 mM) and PTP1 (238 nM) were combined at 30 °C in 100 mM sodium acetate (pH 5.5), 1 mM EDTA; the ionic strength of the buffer was adjusted to I = 0.15 M using NaCl. Aliquots were removed at 1 and 10 min and analyzed by HPLC as described under ``Experimental Procedures.'' Trace A shows Peptide 992N-Bpa, and traces B and C show the peptide after 1 and 10 min of reaction with PTP1, respectively.
In order
to obtain kinetic parameters for the PTP1-catalyzed hydrolysis of
phosphopeptide 992N-Bpa, we employed a continuous spectrophotometric
assay described previously (9) to follow the dephosphorylation
of tyrosine on the peptide. Values of k and K
for PTP1 using peptide 992N-Bpa at pH 6.6 and 30
°C were 69.3 ± 5.9 s
and 3.30 ±
0.51 µM, respectively. These values are similar to those
for the peptide EGFR
(Table 1) and place
peptide 992N-Bpa among the best substrates for PTP1. The kinetic
parameters k
and K
for
peptide 992N-Bpa at pH 8.0 and 30 °C were determined to be 23.4
± 1.9 s
and 9.20 ± 0.75
µM, respectively. Thus the concentrations of photoactive
peptide 992N-Bpa utilized in the cross-linking experiments were well
above that needed for saturation.
To produce peptide
fragments suitable for HPLC analysis, trypsin digestion of the
cross-linked enzyme was carried out. The labeled enzyme was dissolved
in 100 mM NHHCO
and incubated with
trypsin for 24 h at 37 °C (conditions were determined by
proteolysis of unmodified PTP1). A Microcon-10 concentration unit was
then used to remove the trypsin from the reaction. The filtrates
contained approximately 94% of the radioactivity; thus, the majority of
the radioactivity was located within small (molecular weight <
10,000) peptide fragments. The first reaction was analyzed on an
analytical Vydac C18 column (Fig. 3A). Fractions
containing peaks of absorbance at 220 nm were collected and analyzed by
scintillation counting after dissolving 5% of each sample in
scintillation fluid. The major radioactive peak from this experiment
(66% of radioactivity applied to column) eluted at 65.4 min (Peak
A) (Fig. 3A). Minor radioactive peaks in the HPLC
chromatogram (<10% of radioactivity) were present as well, possibly
because multiple points of attachment into peptide chains are possible
with benzophenones(24) , and multiple stereoisomers may be
formed. In addition, multiple peaks may be observed when a single
cross-linked peptide exhibits differential interactions with an HPLC
column(27) . In addition to the labeled digest, two parallel
unmodified digests of PTP1 were carried out under the same conditions.
Both contained a major peak at 81.3 min not present in the modified
digest (Peak B) (Fig. 3B). These results
suggested that Peak B represented a tryptic fragment of PTP1, the
mobility of which was altered upon cross-linking with Peptide 992N-Bpa.
Peak B (
500 pmol, based on the peak height) and the radioactive
peak A (
420 pmol, based on the specific activity) were concentrated in vacuo and analyzed by Edman peptide sequencing.
Figure 3: HPLC analysis of trypsin-digested PTP1. Conditions for the digestions and for HPLC are given under ``Experimental Procedures.'' Absorbance at 220 nm is shown on the y axis. Trace A, trypsin digestion of the complex between Peptide 992N-Bpa and PTP1 analyzed by C18 HPLC. The position of the major radioactive peak is indicated along with its elution time. Trace B, trypsin digestion of unmodified PTP1 analyzed by C18 HPLC. The position of the major peak, which is reduced in trace A is indicated along with its elution time. Elution times of two other peaks are given for comparison to trace A. Trace C, trypsin digestion of the complex between Peptide 992N-Bpa and PTP1 analyzed by C4 HPLC. The position of the major radioactive peak is indicated along with its elution time. Trace D, trypsin digestion of unmodified PTP1 analyzed by C4 HPLC.
The
second preparative cross-linking reaction was carried out under
conditions similar to the first reaction. Again, >94% of the
radioactivity was recovered in the Microcon-10 filtrate after trypsin
digestion. In this case, radioactive tryptic fragments were separated
by another method, reverse-phase HPLC using an analytical C4 column.
The major peak of radioactivity in this experiment (63% of total
radioactivity applied to column) eluted at 75.6 min (Peak C) (Fig. 3C). For comparison, a trypsin digest of
unmodified PTP1 was carried out as described above and analyzed by HPLC
on a C4 column. The peak at 75.6 min was substantially smaller in the
unmodified digest, suggesting that Peak C arose by cross-linking of
Peptide 992N-Bpa to a tryptic fragment (Fig. 3D). Peak
C (350 pmol, based on the specific activity) was concentrated and
sequenced under the same conditions as Peaks A and B as described
above.
The sequences of the peptides obtained in this manner are
given in Table 2. All three sequenced peptides correspond to the
sequence of PTP1 in the region Ala-Arg
.
Sequencing terminated after the cycle corresponding to
Arg
, consistent with the end of a tryptic fragment. For
Peak B, which arose from the tryptic digest of native PTP1, an
additional peptide was present at a lower amount. For Peaks A and C, a
low recovery of phenylthiohydantoin-derivative (approximately 10% of
the yield compared with the previous cycle) was observed in the 11th
cycle (corresponding to Ile
). These results suggest that
the covalent modification took place at Ile
(see
``Discussion''). At each sequencing cycle during the analysis
of the radioactive peaks, one-half of the
phenylthiohydantoin-derivative was identified by HPLC and the remainder
analyzed by scintillation counting. In no case was any radioactivity
observed to be released from the sequencing filter in these
measurements. Instead, the radioactivity remained associated with the
filter (as judged by scintillation counting), even after complete
sequencing of the peptide fragments. This result may reflect the
hydrophobic nature of the cross-linked adduct between Ile
and benzoyl-Phe.
When bound to a protein, synthetic peptides containing the photoactive amino acid analog Bpa cross-link efficiently with nearby residues upon photoactivation(23) . Benzophenone-type labels such as Bpa offer several advantages over other photoaffinity reagents (22, 23, 24) . Benzophenones are activated at 350 nm, avoiding lower wavelengths which are potentially damaging to proteins. The photolytically generated intermediate reacts preferentially with C-H bonds, even in the presence of aqueous buffer; hence, normally unreactive amino acid side chains may be modified in a binding site. Finally, the cross-linked product is stable to proteolysis and to other manipulations involved in characterizing the labeled site. Because of these features of benzophenone photoaffinity labels, the yields of cross-linked products are often quite high, and they have been successfully applied in a wide variety of biochemical systems, including protein kinase active sites (21, 23) and SH2 domains(26, 28) . Based on photochemical studies using model benzophenone-containing compounds, the reactive volume of Bpa may be approximated as a sphere with a radius of 3.1 Å centered on the ketone oxygen(24) . Cross-linked regions of the target protein may be considered to be within this distance of the bound peptide at the point of photoactivation.
In the present study a good substrate for rat
PTP1 (k/K
of the parent
peptide EGFR
= 2.88
10
M
s
;(17) )
has been substituted with Bpa at the position immediately
NH
-terminal to phosphotyrosine. This position was chosen
based on the results of Ala scanning experiments, which demonstrate
that amino acid residues NH
-terminal to phosphotyrosine are
important for binding and catalysis, especially at the -1
position (10) . PTP1 was relatively tolerant of amino acid
changes at the -1 position (relative to phosphotyrosine). Thus, a
Glu to Ala substitution at this position increased K
by a factor of 4.7-fold and reduced k
from
75.7 s
to 54.8 s
for PTP1. A
comparable change produces a 126-fold drop in k
/K
for the Yersinia PTPase(10) . Although this may be due to the absence of
the negatively charged glutamate, the dramatic drop in k
/K
may also be a
consequence of the inability of the alanine moiety to interact strongly
with the enzyme surface in a favorable fashion. The negatively charged
residues at the -1 position may be obligatory for peptide
recognition by some PTPases but dispensable for others. Indeed,
phosphopeptide 992N-Bpa is an excellent substrate for PTP1 with k
/K
comparable with the
parent peptide EGFR
(Table 1). This is a
surprising result, since the molecular properties of Bpa differ
drastically from Glu.
Because 992N-Bpa contains a phosphotyrosine
residue and is a good substrate for PTP1, the photocross-linking
experiments were conducted at pH 8.0 and at 4 °C to reduce the rate
of substrate hydrolysis (10, 18) . The conditions for
the photoinactivation were comparable with those for the
crystallization of the PTP1B C215S-phosphopeptide complex which were at
pH 7.5 and 4 °C(14, 19) . The pseudo first order
rate constant for photoinactivation was 0.033 s under these conditions. PTP1 catalysis is 5.8-fold slower at 3.5
°C than at 30 °C(18) . Since k
for 992N-Bpa hydrolysis was measured to be 23.4 s
at pH 8.0 and 30 °C, the value of k
is
estimated to be 4.0 s
at 4 °C and pH 8. Thus,
the rate for substrate turnover was 120-fold faster than the rate for
photoinactivation. At pH 5.5, photoinactivation did not occur at a
significant rate (data not shown). This was presumably because of the
even faster dephosphorylation of the label (k
= 75 s
at pH 5.5 versus 23.4
s
at pH 8). When the phosphate group is removed from
a phosphotyrosine-containing peptide, the resulting dephosphopeptide
does not bind to PTPases(9) . This may explain the low
photocross-linking efficiency seen at pH 5.5.
The Bpa-containing
peptide cross-linked to a helical region in the NH terminus
of PTP1 in two separate experiments. The
-helix modified by
Peptide 992N-Bpa (designated
2` in Barford et
al.(14) ) lies near the active site cleft of PTP1 (Fig. 4). However, this helix is situated on the opposite side
of the cleft from Arg
, the residue in the Cys
Ser mutant of PTP1B that makes contact with the bound
peptide Asp-Ala-Asp-Glu-pTyr-Leu-NH
at the -1 and
-2 positions in the cocrystal structure(19) . Arg
is located on a loop that connects
1 and
1 in the PTP1B
structure. In the complexed structure, the NH
-terminal
portion of the peptide adopts a twisted
-strand conformation, and
amino acids at the -4 through +1 positions make contact with
the enzyme. The recognition pocket for phosphotyrosine represents the
dominant driving force for peptide binding since phosphotyrosine
contributes about 53% of the peptide solvent-accessible surface
area(19) . Invariant nonpolar residues in the PTPase catalytic
domains (Tyr
, Val
, Phe
,
Gln
) form the binding site for the phenyl ring of
phosphotyrosine. The phosphoryl group in phosphotyrosine is surrounded
by residues corresponding to the PTPase signature motif(4) .
This suggests that the mechanism for phosphotyrosine recognition is
similar among all PTPases. On the other hand, Arg
,
Ile
, and the residues of helix
2` are not conserved
among PTPases; specific recognition of substrates may be accomplished
by residues which are variable. The side chain of Asp
in
PTP1 forms two hydrogen bonds between the main chain nitrogens of
phosphotyrosine and the +1 residue, which are important for
stabilizing the substrate peptide conformation. The guanidinium side
chain of residue Arg
forms salt bridges with the side
chains of Glu and Asp residues on the peptide (at positions -1
and -2, respectively), suggesting an important role in peptide
recognition(19) . Leucine at the +1 position in the
phosphotyrosyl peptide makes van der Waals contacts with
Val
, Ile
, and Gln
of the
enzyme. In that structure, the side chain of residue Arg
interacts with the carbonyl oxygen of the leucine residue at the
+1 position through a bound water molecule. No additional
interactions between helix
2` and the bound peptide are present in
the co-crystal structure(19) .
Figure 4:
Crystal structure of PTP1 complexed with
tungstate. A ribbon diagram of the PTP1 structure (14) was
created using the program MOLSCRIPT(30) . Tungstate ion is
shown in Corey-Pauling-Koltun format. The positions of residues
Arg and Ile
(in helix
2`) are
indicated.
Photocross-linking of Peptide
992N-Bpa to helix 2` implies that the peptide is bound to the
enzyme active site in a different conformation than that observed for
Asp-Ala-Asp-Glu-pTyr-Leu-amide in the crystal structure. Since the
photoaffinity label-containing phosphopeptide acts as a good substrate
for the enzyme, it must consequently be bound in the active site in a
conformation that is compatible with PTP1 catalytic function. One
possible alternative peptide binding mode would be if Peptide 992N-Bpa
binds in the opposite orientation to that seen in the crystal
structure, which could place the P-1 Bpa residue closer to
Ile
. Replacement of the negative charge at position
-1 in Peptide 992N-Bpa by a bulky hydrophobic Bpa residue may in
fact reduce the interaction with the side chain of Arg
to
such an extent that the mode of binding seen in the crystal structure
is no longer energetically favored. Thus, interaction with helix
2` may be important in substrates of PTP1 which do not contain an
acidic residue NH
-terminal to tyrosine. Interestingly,
2` in PTP1 is composed of amino acid residues
Trp-Ala-Ala-Ile-Tyr-Gln-Asp-Ile-Arg-His-Glu
which are fairly hydrophobic. Furthermore, in enzymes that are
particularly dependent on an acidic residue at the -1 position (e.g. Yersinia phosphatase), helix
2` may not be an
important determinant for peptide recognition. This hypothesis is
supported by the fact that Peptide 992N-Bpa photocross-links only
weakly to the Yersinia enzyme. (
)An alternative
explanation for the results would be that 992N-Bpa binds in a similar
fashion as observed by Jia et al.(19) , but that the
entire NH
terminus of PTP1, which contains the
2`
helix, moves toward the bound peptide.
Although rat PTP1 and its human homolog PTP1B are localized to the cytoplasmic side of endoplasmic reticulum(5, 29) , which may provide a level of regulation, in vitro they are not very specific PTPases because they dephosphorylate a wide variety of substrates. As shown in the three-dimensional structure of the catalytic domain of human PTP1B(14) , the protein surface surrounding the catalytic cleft is relatively open and consists of a number of depressions and protrusions. This may allow numerous modes of peptide recognition and is consistent with the ability of PTP1 to hydrolyze a wide variety of phosphotyrosine-containing substrates with nearly equal efficiency (9, 10, 17; this study). It is reasonable to suggest, based on both kinetic and structural studies, that binding interactions between PTP1 and the side chain of tyrosine, the phosphate group, the main chain nitrogens of phosphotyrosine and the +1 residue are conserved for all peptide substrates. The orientation of individual peptide/protein substrates may be different from each other and may be dictated by specific interactions between amino acid side chains in the vicinity of phosphotyrosine and residues near the enzyme active site cleft. Using photoaffinity labeling techniques, we have identified a unique mode of substrate recognition by PTP1, which is distinct from that observed in the crystal structure with a different peptide. These observations have implications for the design and development of PTPase inhibitors; they indicate that a systematic investigation of amino acid specificity at sites in close proximity to the phosphotyrosine may reveal sequences that are preferentially recognized by PTPases.