(Received for publication, June 27, 1995; and in revised form, September 22, 1995)
From the
Yersinia protein-tyrosine phosphatase substrates have
been synthesized employing an expedient methodology that incorporates
phosphorylated non-amino acid residues into an active site-directed
peptide. While the peptidic portion of these compounds serves an enzyme
targeting role, the nonpeptidic component provides a critical
assessment of the range of functionality that can be accommodated
within the active site region. We have found that the Yersinia phosphatase hydrolyzes both L- and D-stereoisomers of phosphotyrosine in active site-directed
peptides, with the former serving as a 10-fold more efficient substrate
than the latter. In addition, this enzyme catalyzes the hydrolysis of a
variety of aromatic and aliphatic phosphates. Indeed, a peptide bearing
the achiral phosphotyrosine analog, phosphotyramine, is not only the
most efficient substrate described in this study, it is also one of the
most efficient substrates ever reported for the Yersinia phosphatase. Straight chain peptide-bound aliphatic phosphates of
the general structure,
(Glu)-NH-(CH
)
-OPO
(n = 2-8), are also hydrolyzed, where the most
efficient substrate contains seven methylene groups. Finally, a
comparison of the substrate efficacy of the peptide-bound species with
that of the corresponding non-peptidic analogs, reveals that the
peptide component enhances k
/K
by up to
nearly 3 orders of magnitude.
Protein-tyrosine phosphatases (PTPases) ()are
emerging as essential regulators of a variety of fundamental cellular
processes such as cell growth, mitogenesis, metabolism, gene
transcription, cell cycle control, and the immune
response(1, 2, 3) . Together with the
protein-tyrosine kinases (PTKs), the PTPases control the state of
tyrosine phosphorylation on cellular proteins. PTPases constitute a
growing family of enzymes that rival PTKs in terms of structural
diversity and complexity. Unlike tyrosine-specific and
serine/threonine-specific kinases, which share conserved sequences in
their catalytic domains, PTPases show no sequence similarity with
serine/threonine phosphatases, or the broad-specificity phosphatases
such as acid or alkaline phosphatases(4, 5) . However,
all members of the PTPase subfamily, from bacteria to mammals, do share
a strong sequence similarity within a 250 residue span of the catalytic
domain(6) .
Despite a rapidly growing appreciation of the biological importance of PTPases in signal transduction, a detailed understanding of their substrate specificity is lacking. How do PTPases distinguish between the diversity of phosphoproteins that they encounter within the cell? Studies using synthetic phosphopeptides have demonstrated that PTPases are sensitive to the amino acid sequence that encompasses the phosphotyrosine moiety(7, 8, 9, 10, 11) . In addition, it is clear that the phosphotyrosine residue itself is absolutely essential for PTPase recognition of protein-based substrates as well as peptide-based substrates that are designed to mimic the former. For example, PTPases do not bind tyrosine-bearing peptides that lack the phosphate functionality(9, 12) . Furthermore, replacement of the critical phosphotyrosine residue with an O-methylated phosphotyrosine generates a totally inert derivative(9) . Finally, PTPases appear to exhibit a strict bias for phosphotyrosine since they do not utilize phosphoserine or phosphothreonine as substrates in protein- and peptide-based environments(13, 14, 15) . Such discriminatory behavior is not surprising given the difference in orientation and distance of the phosphate moiety, relative to the adjacent peptide backbone, in phosphotyrosine compared to those in phosphoserine/phosphothreonine. This certainly implies that a tyrosine moiety, in conjunction with the negatively charged phosphate group, is crucial for PTPase recognition. Based upon these structural considerations, several nonhydrolyzable analogs of phosphotyrosine have been prepared and inserted into PTPase-targeted peptides. For example, an aromatic moiety and a negatively charged phosphonate are contained within phosphonomethyl phenylalanine, a species that lacks a hydrolyzable aromatic ester. Indeed, phosphonomethyl phenylalanine-containing peptides have been shown to be effective, reversible inhibitors of PTPases(11, 16, 17) .
In spite of the fact that the phosphotyrosine moiety appears to be essential for PTPase recognition of peptide- and protein-based systems, a number of small, non-peptidic residues have been found to serve as reasonably efficient PTPase substrates. In this regard, the most widely utilized substrate is p-nitrophenyl phosphate. In addition, we have recently demonstrated that PTPases will also catalyze the dephosphorylation of a variety of alkyl phosphates, including the individual amino acids phosphoserine and phosphothreonine(18) . However, these substrates are not contained within the context of a protein-like environment, a structural framework that could assist in targeting specific PTPases. We have recently devised a chemical strategy that fuses peptidic and nonpeptidic components into compounds that act as unusually efficient substrates for protein kinases (19, 20, 21) . These fusion compounds have provided a critical assessment of the range of functionality that can be accommodated within the active site of protein kinases, information that should prove to be of decided assistance in the design of potent inhibitory agents. In addition, these amalgamated species have been utilized to distinguish between kinases that are otherwise indistinguishable with conventional peptide substrates(22) . We now report that this strategy, for the first time, has been successfully applied to a member of the protein phosphatase family. We have constructed a structurally diverse array of phosphorylated peptide-aminoalcohol fusion compounds to assess the active site substrate specificity of the Yersinia PTPase. This phosphatase is required for the pathogenicity of Yersinia pestis, the bacterium responsible for the bubonic plague, also known as the Black Death(23) . In contrast to the currently held beliefs that phosphotyrosine is absolutely essential for PTPase recognition of protein- and peptide-based substrates, we have found that the Yersinia PTPase will catalyze the hydrolysis of a wide variety of both aromatic and aliphatic phosphates in peptides. This not only greatly expands the range of compounds that can be recognized by PTPases, but also provides a mechanism for utilizing the specificity inherent within the peptide component of these compounds to target specific PTPases.
All chemicals were purchased from Aldrich, except for
piperidine and protected amino acid derivatives (Advanced Chem Tech)
and the Fmoc-Glu(O-t-butyl)-2-methoxy-4-alkoxybenzyl
alcohol resin (Peninsula Laboratories, Inc.). The structure of all new
compounds were confirmed by H NMR (300 Mhz),
C
NMR (22.5 Mhz),
P NMR (161.9 Mhz), and fast atom
bombardment mass spectral analyses (positive and negative ion). Enzyme
assay solutions were prepared with deionized/distilled water.
Figure S1:
Scheme 1Synthesis of the
peptide-based Yersinia PTPase substrates. Aminoalcohols were
protected at the amine moiety with Fmoc-succinimide and subsequently
phosphitylated to furnish B. The phosphite was oxidized to the
phosphotriester and then deprotected to yield C. This species was
coupled to a side chain-protected tetrapeptide, and then all protecting
groups were removed with trifluoroacetic acid. The tetraglutamic acid
peptide was obtained via standard solid phase peptide synthesis and was
cleaved from the 2-methoxy-4-alkoxybenzyl alcohol resin with side chain
protecting groups intact using 1% trifluoroacetic acid in
CHCl
. See ``Materials and Methods''
for the preparation of peptides 1 and 4 and the free amine
6.
Kinetic parameters k and K
were determined by analyzing the experimental data through a
nonlinear least-squares fit algorithm of this equation, where k
is the catalytic turn-over number, K
is the Michaelis constant, E
is the enzyme concentration, and p and p
are the product concentrations at time t and infinity, respectively.
An in-depth analysis of the active site substrate specificity
of PTPases may furnish critical information that could assist in the
design of potent active site-directed inhibitors. For example, such an
analysis can provide detailed structural data regarding the range of
functionality that can be readily tolerated by the active site of these
enzymes. In this regard, PTPases, such as the phosphatase isolated from Yersinia, are known to utilize a variety of low molecular
weight aromatic and aliphatic phosphates as substrates. Unfortunately,
these rather simple compounds lack the protein-like environment that
may be crucial for targeting individual PTPases in a highly specific
fashion. Clearly, peptide-based species offer one possible solution to
the issue of specificity. In order to take advantage of this
specificity, we have attached an array of aliphatic and aromatic
phosphates to the C terminus of an active site-directed peptide. Acidic
amino acid residues on the amino-terminal side of phosphotyrosine are
important recognition elements for the Yersinia PTPase(8, 11) . Consequently, we prepared the
peptide (Glu)-Tyr(P)-NH
. This species is
hydrolyzed by Yersinia PTPase (pH 6 and 30 °C) with k
and K
values of (1.2
± 0.1)
10
s
and 0.31
± 0.06 mM, respectively (Table 1). For
comparative purposes, the K
and V
values associated with the Yersinia PTPase-catalyzed
dephosphorylation of Asp-Ala-Asp-Glu-Tyr(P)-NH
and
epidermal growth factor receptor
(Asp-Ala-Asp-Glu-Tyr(P)-Leu-Ile-Pro-Gln-Gln-Gly) are provided in Table 1(11) . Clearly, (Glu)
-Tyr(P)-NH
is a useful structural starting point for the preparation of
peptide-based analogs in which the phosphotyrosine residue is replaced
with various phosphorylated aminoalcohols. Based upon these results, we
prepared peptide-based PTPase substrates of the general formula,
Glu-Glu-Glu-Glu-NH-R-OPO
(Fig. S1). The amine portion of an aminoalcohol (A) was
first protected as the Fmoc-derivative and the alcohol subsequently
phosphitylated to furnish B(24, 25) . This
species was then sequentially oxidized to the phosphotriester and the
Fmoc group removed under basic conditions to yield the free amine C. The latter was readily coupled to the Bop-activated form of
the protected tetrapeptide,
Boc-Glu(O-t-butyl)-Glu(O-t-butyl)-Glu(O-t-butyl)-Glu(O-t-butyl)-COOH
and all the protecting groups subsequently removed with 95%
trifluoroacetic acid, 5% water to furnish the desired peptide-based
phosphorylated aminoalcohol D.
We first determined whether
the Yersinia PTPase activity is sensitive to stereochemistry
at the -carbon of the phosphotyrosine residue.
(Glu)
-D-Tyr(P)-NH
(4) exhibits
a k
of 1.3
10
s
and a K
of 3.0 mM. The resulting k
/K
is 9-fold smaller than
that of (Glu)
-Tyr(P)-NH
(1), which is
exclusively due to an order of magnitude increase in K
for the D-phosphotyrosine-bearing peptide.
Interestingly, PTPases catalyze the hydrolysis of free D-phosphotyrosine and L-phosphotyrosine with equal
efficiency(29) . In contrast, it is clear that the Yersinia PTPase prefers the naturally occurring L-stereoisomer
when the phosphorylated amino acid is constrained within a
peptide-based environment. Will the Yersinia PTPase recognize
achiral residues substrates within such an environment? To address this
question, we prepared
(Glu)
-NH-(CH
)
C
H
-OPO
(5) via the synthetic scheme outlined above. The
phosphotyramine residue contained within this peptide lacks
the
-carboxamide moiety present in phosphotyrosine (1). Surprisingly, the achiral derivative is a 5-fold more
efficient substrate (in terms of k
/K
) than its naturally
occurring chiral counterpart. This is a consequence of a sharp drop in K
for
(Glu)
-NH-(CH
)
C
H
-OPO
(5). Peptide 5 is not only the best substrate described
in this study, it is also more efficiently hydrolyzed than
Asp-Ala-Asp-Glu-Tyr(P)-Leu-NH
and is nearly as
efficiently hydrolyzed as the best substrate reported for Yersinia PTPase, Asp-Ala-Asp-Glu-Tyr(P)-Leu-Ile-Pro-Gln-Gln-Gly (Table 1) (11) . Is the achiral phosphorylated tyramine
moiety exceptionally reactive? We prepared the peptide-free derivative
NH
-(CH
)
C
H
-OPO
(6) in order to address this question. As expected, 6 behaves as a typical aryl phosphate. The k
(0.32
10
s
) and K
(3.2 mM) for this species are nearly
identical to those exhibited by p-nitrophenyl phosphate (k
(0.35
10
s
) and K
(2.6 mM))
under similar conditions(6) . These results reveal two
significant facets of the substrate specificity of the Yersinia PTPase. First, in the absence of amino acid residues on the
carboxyl-terminal side of phosphotyrosine, substituents at the
-position of the phosphoresidue interfere with PTPase activity.
Second, a peptide moiety can dramatically enhance the substrate
efficacy of a simple aromatic phosphate, such as phosphotyramine (a
224-fold enhancement in k
/K
of
(Glu)
-NH-(CH
)
C
H
-OPO
(5) versus
NH
-(CH
)
C
H
-OPO
(6)).
In addition to the influence exerted by
stereochemistry, we have found that the distance between the peptide
backbone and the aromatic phosphate moiety also controls the efficacy
of PTPase-catalyzed hydrolysis. When the distance between the peptide
unit and the aryl phosphate group is shortened by a single methylene
unit [i.e. compound
(Glu)-NH-CH
C
H
-OPO
(7)] an 18-fold reduction in k
/K
occurs relative to that
observed with
(Glu)
-NH-(CH
)
C
H
-OPO
(5). The drop in substrate efficacy is primarily due to an
8-fold increase in K
(Table 1). However, a
minor 2.4-fold decrease in k
is observed as
well. In addition, we prepared an analog of
(Glu)
-NH-CH
C
H
-OPO
,
one which contains a methylene group between the aromatic ring and the
phosphate moiety (i.e. (Glu)
-NH-CH
C
H
CH
-OPO
(8)). In this case, the distance between the phosphate and the
peptide backbone is similar to that present in
(Glu)
-NH-(CH
)
C
H
-OPO
(5). However, the phosphate moiety is no longer directly
attached to the aromatic ring. Compound 8 does serve as a
substrate for the Yersinia PTPase, an observation consistent
with our earlier results that demonstrated that alkyl phosphates are
hydrolyzed by tyrosine-specific phosphatases(18) . However,
compared to its structural isostere 5, compound 8 exhibits a 150-fold smaller k
and a 30-fold
larger K
. Clearly, the Yersinia PTPase is
more efficient in processing the aryl phosphate in
(Glu)
-NH-(CH
)
C
H
-OPO
(5) than its benzylic counterpart in
(Glu)
-NH-CH
C
H
CH
-OPO
(8). However, it is not clear if this is a consequence of some
intrinsically lower chemical reactivity associated with the benzyl
phosphate moiety or if this is due to an altered active site-bound
orientation of the phosphate group induced by the nature of the
aromatic ring substituents. The former possibility is the most likely
since alkyl phosphates are hydrolyzed at a significantly reduced rate
compared to their aromatic counterparts ( (18) and see below).
Finally, we note that
(Glu)
-NH-CH
C
H
CH
-OPO
(8) is a significantly (46-fold) more efficient substrate than
pyridoxal 5`-phosphate(18) , a non-peptidic species that
contains a benzylic phosphate moiety. This comparison once again
illustrates the importance of the peptide component and demonstrates
that interactions removed from the site of bond cleavage and formation
can be beneficial for catalysis.
Barford et al.(30) recently proposed that PTPase specificity for
phosphotyrosine-containing peptides probably results from the depth of
the active site cleft since the smaller phosphoserine and
phosphothreonine side chains should be unable to reach the phosphate
binding site. Since simple alkyl phosphates can be processed by PTPases
(although not as efficiently as aryl phosphates)(18) , and
since the presence of a peptide enhances the substrate reactivity ( (11) and see above), we expected that a peptide-linked alkyl
phosphate should make a reasonable PTPase substrate. This should
especially be the case if the distance between the peptide backbone and
the phosphate moiety corresponds to that present in a peptide-linked
phosphorylated tyrosine. Such an ``optimal'' distance should
approximately be six CH units. We prepared a series of
peptide-based phosphorylated aliphatic alcohols (Table 2) in
order to explore the validity of this supposition.
The k/K
for the Yersinia PTPase-catalyzed hydrolysis of
(Glu)
-NH-(CH
)
-OPO
(9) is 0.20 mM
s
(Table 2). Although this value is significantly lower than
all of the aromatic phosphates illustrated in Table 1, peptide 9 is a considerably better substrate than its non-peptidic
counterpart,
NH
-(CH
)
-OPO
(18) . Indeed, the hydrolytic turnover of the latter is so slow
that we could not accurately measure the kinetic constants. However,
since we were able to obtain a k
(1.8
s
) and K
(48 mM) for DL-
-glycerophosphate(18) , we have attributed the
lack of activity of
NH
-(CH
)
-OPO
to the positively charged
-substituent. The specific activity of
the Yersinia PTPase at 20 mM substrate concentration
toward
NH
-(CH
)
-OPO
is 149-fold lower than DL-
-glycerophosphate. Since the K
values for these simple aliphatic alkyl
phosphates are significantly higher than 20 mM, the specific
activity measurement for these substrates at this concentration
provides an assessment of the kinetic parameter k
/K
. To a first
approximation, the k
/K
for
NH
-(CH
)
-OPO
is 2.6
10
mM
s
. Consequently,
(Glu)
-NH-(CH
)
-OPO
(9), which lacks the positively charged
-substituent and
contains an appended active site-directed peptide, is approximately
770-fold more efficiently hydrolyzed than O-phosphorylethanolamine.
We next addressed the issue of
optimal distance between the phosphate moiety and the peptide backbone (Table 2). As the number of methylene groups n in
(Glu)-NH-(CH
)
-OPO
increases from 2 up through 5 (compounds 9-12), an overall
10-fold increase in k
/K
is
apparent. Most of the improvement in the k
/K
parameter is a
consequence of enhanced k
values (Table 2). However, this improvement accelerates rapidly at n = 6 and peaks at n = 7. The 26-fold
enhancement in k
/K
for
(Glu)
-NH-(CH
)
-OPO
(13) and 70-fold enhancement in substrate efficacy for
(Glu)
-NH-(CH
)
-OPO
(14) (both relative to
(Glu)
-NH-(CH
)
-OPO
(9)) are due to substantial improvements in the k
parameter. However, the catalytic efficiency
of compound 15 (n = 8) is more than an order of
magnitude less than its n = 7 counterpart (Table 2). These results are consistent with the hypothesis that
the optimal distance between the phosphate moiety and the peptide
backbone corresponds to the length of a tyrosine side chain. One
possible explanation for the ability of peptides containing a
shorter-than-ideal chain length to serve as substrates may be due to
the insertion of a portion of the peptide backbone into the enzyme
active site. Similarly,
(Glu)
-NH-(CH
)
-OPO
(15), which possesses a longer-than-ideal chain length, may be
able to position itself somewhat further from the active site, which
would allow the phosphate moiety, on the relatively long side chain, to
interact with the catalytic apparatus in a favorable fashion. In short,
the peptide may be able to ``slide'' along the enzyme surface
until the phosphate is properly positioned in the active site. We have
applied a similar type of reasoning to explain the relatively broad
active site substrate specificity of
pp60
(21) . Finally, we prepared the
phosphate monoester of the cyclohexyl derivative 16 (Table 2). The efficacy of this peptide as a substrate is
similar to species 11 (n = 4) and 12 (n = 5), compounds in which the phosphate is fixed
at a similar distance from the peptide backbone. However, this result
is surprising in light of the fact that phosphorylated secondary
alcohols are generally much poorer substrates than their primary
alcohol-containing counterparts (18) . At this point, we do not
know whether the favorable kinetics associated with 16 is due to
the presence of the peptide unit or a consequence of unusual reactivity
associated with the cyclohexyl group.
PTPases are much more
effective catalysts for the hydrolysis of aryl phosphates than alkyl
phosphates. For example, the Yersinia PTPase dephosphorylates
aryl phosphates, such as p-nitrophenyl phosphate, 2-3
orders of magnitude more rapidly than alkyl phosphates of primary
alcohols(18) . The Yersinia PTPase also catalyzes the
exchange reaction between O-labeled phosphate and solvent
water(31) . Similarly, the k
value for
exchange at pH 6.0 is 350-fold slower than the k
value for the Yersinia PTPase-catalyzed hydrolysis of p-nitrophenyl phosphate. The PTPase-catalyzed reaction
involves a phosphoenzyme
intermediate(32, 33, 34) . The formation of
the phosphoenzyme intermediate involves attack by the active site
cysteine on the phosphorus atom and subsequent release of the leaving
group (e.g. phenoxide, alkoxide, or hydroxide). Since the
pK
values of the conjugate acid of the leaving
group (alkoxide (RO
) for alkyl phosphate and
hydroxide (HO
) for inorganic phosphate) are both
approximately 15-16, the repulsion of alkoxides or a hydroxide
would require much greater assistance from the enzyme than phenoxides
(which have pK
values typically below 10). The
phosphorylated aliphatic alcohols in this study should all have leaving
group pK
values of 15-16. In short, their
intrinsic chemical reactivity should be similar to simple alkyl
phosphates. Consequently, it is remarkable that
(Glu)
-NH-(CH
)
-OPO
exhibits a turnover number of 25 s
, which is only
14-fold slower than p-nitrophenyl phosphate. This is another
clear example that binding interactions substantially removed from the
site of bond cleavage and formation are utilized to facilitate
catalysis.
We also performed a preliminary analysis of the binding
affinity of two phosphorylated aliphatic alcohol peptides to the Yersinia PTPase. We measured the ability of peptides 13 (n = 6) and 15 (n = 8) to
inhibit the enzyme-catalyzed hydrolysis of p-nitrophenyl
phosphate. Both
(Glu)-NH-(CH
)
-OPO
and
(Glu)
-NH-(CH
)
-OPO
serve as competitive inhibitors versus the phosphomonoester
substrate, with K
values of 360 ± 88 and
960 ± 260 µM, respectively. As a comparison, DL-
-glycerophosphate competitively inhibits the Yersinia PTPase-catalyzed hydrolysis of p-nitrophenyl
phosphate at pH 7.0 with a K
of 20.4 ± 5.6
mM(18) . Therefore, we conclude that the presence of
the peptide moiety does contribute to additional substrate binding.
This additional binding energy may be utilized to position the
catalytic groups in the enzyme active site to efficiently carry out the
chemical steps.
In summary, we have prepared an array of structurally diverse phosphorylated aminoalcohols and have attached these to the carboxyl terminus of Glu-Glu-Glu-Glu. These amalgamated peptide-aminoalcohol phosphates have been utilized to probe the active site specificity of the Yersinia PTPase. We have found that efficient binding and catalysis by the Yersinia PTPase is dependent upon the distance between the phosphate moiety and the peptide main chain backbone. Although substrate efficacy is distance-dependent, this enzyme does hydrolyze a wide variety of aliphatic and aromatic phosphates. In addition, when compared with the catalytic efficacy of non-peptidic analogs, it is clear that the peptide component of these amalgamated substrates significantly contributes to efficient enzyme-catalyzed hydrolysis. For example, ethanolamine phosphate is hydrolyzed nearly 3 orders of magnitude more effectively when attached to the active site directed peptide Glu-Glu-Glu-Glu. The strategy that we have developed provides the opportunity to deliver relatively simple functionality to specific PTPases through the use of appropriate peptide templates. Furthermore, a comparative analysis of the active site specificities of protein phosphatases may reveal key differences in the ability of individual enzymes to tolerate specific structural motifs. Clearly, any observed differences should prove useful in the design of PTPase-specific inhibitors. The latter would be of decided benefit in helping to define the role of PTPases in cellular signaling.