(Received for publication, June 1, 1995; and in revised form, July 17, 1995)
From the
A potential distinguishing feature between protein tyrosine
kinases and homologous serine/threonine kinases is the function of the
catalytic base in these enzymes. In this study, we show that a peptide
containing the unnatural amino acid trifluorotyrosine shows remarkably
similar efficiency as a substrate of the tyrosine kinase Csk
(C-terminal Src kinase) compared with the corresponding
tyrosine-containing peptide despite a 4-unit change in the phenolic
pK. These results argue against the
importance of early tyrosine deprotonation by a catalytic base in Csk.
To further explore the role of the proposed catalytic base, the Csk
mutant protein D314E was produced. This mutant displayed a significant
reduction in k
(approximately 10
)
but relatively little effect on substrate K
values compared with wild-type Csk. Examination of the thio
effect (k
-ATP/k
-adenosine
5`-O-(thiotriphosphate)) for D314E Csk led to the suggestion
that a role of aspartate 314 may be to enhance the reactivity of the
-phosphate of ATP toward electrophilic attack. These results may
have significant impact on protein tyrosine kinase inhibitor design.
Protein tyrosine kinases play central roles in cell proliferation, cell differentiation, and signaling processes(1) . While their enzymatic activity, transfer of a phosphoryl group from ATP to tyrosines in proteins, was first described over 15 years ago, details of tyrosine kinase catalytic mechanism have largely remained elusive. In contrast, their homologous family member protein serine/threonine kinases have been more intensively studied and are better characterized enzymologically(2, 3, 4, 5, 6, 7, 8) . In order to rationally develop specific inhibitors for these enzymes (9) as well as to delineate the biochemical consequences of their in vivo regulation, a more complete understanding of tyrosine kinase catalysis is needed.
Recently, we carried out a
preliminary mechanistic evaluation of the protein tyrosine kinase Csk
(C-terminal Src kinase) from humans(10) , overproduced in and
purified from Escherichia coli(11) . It was shown
that, like the better studied serine/threonine kinase protein kinase A,
the Csk-catalyzed kinase reaction obeys a ternary complex mechanism,
most likely with rapid and random binding of the substrates ATP and 4:1
poly(Glu,Tyr). This argued against a covalent phosphoenzyme
intermediate. Moreover, using ATPS (
)and microviscosity
effects, it was shown that in the Csk ATP reaction, product ADP
diffusional release is partially rate-determining for the overall
reaction(10) . The lack of a large deuterium solvent kinetic
isotope effect (actually a slightly inverse, k
H/k
D = 0.74, was
seen) in the ATP
S reaction (where the chemical step appeared fully
rate-determining) suggested that tyrosine hydroxyl deprotonation is
asymmetric in the reaction transition state. Because a
tetrafluorotyrosine-containing peptide was previously reported to be a
tight binding inhibitor (and not detectably a substrate) for insulin
receptor tyrosine kinase(12) , a phenoxide anion was reasoned
to be transition state like(10) . The phenoxide anion was
proposed to be the species attacking the
-phosphate of ATP (see Fig. 1). In principle, the role of a catalytic base may be a
mechanistically distinguishing feature between serine/threonine and
tyrosine kinases because the pK
values of
tyrosine hydroxyls (approximately 10) are significantly lower than
those of serine or threonine hydroxyls (approximately
13-15)(2, 3, 4, 5, 7, 8) .
Figure 1: Early tyrosine deprotonation in Csk catalysis.
To further explore the role of a proposed active site
catalytic base, we decided to pursue studies on Csk using an unnatural
fluorotyrosine-substituted peptide. The goal was to determine the
inhibitory properties toward Csk of an isosteric tyrosine analog with a
reduced pK. Using tyrosine
phenol-lyase(13, 14) , trifluorotyrosine was generated
(see Fig. 2A) and after conversion to the Fmoc
derivative (17) incorporated into the Src autophosphorylation
peptide EDNE(F
Y)TA by solid phase peptide synthesis.
EDNEYTA has previously been reported to be a Csk
substrate(18) , and we confirmed that here (k
= 14 ± 0.4 min
, K
= 6.1 ± 0.4 mM, see Fig. 2D). The enzymatically synthesized
trifluorotyrosine was presumed enantiomerically pure (with L-configuration) and was measured by
spectrophotometric titration to have a phenolic pK
of 6.2 ± 0.3. The pK
of the
trifluorotyrosine in EDNE(F
Y)TA was found to be 6.5
± 0.2. These pK
values are approximately 4
units below that of standard tyrosine. Although they are marginally
higher than the pK
measured for
tetrafluorotyrosine (pK
= 5.4) (19) used for study with the insulin receptor tyrosine kinase,
the pK
of 6.5 implies that approximately 90% of
EDNE(F
Y)TA exists as the phenoxide anion at pH 7.4 (the
enzyme assay pH).
Figure 2:
Trifluorotyrosine synthesis, inhibitory
properties, and substrate behavior with Csk. A, synthesis of
trifluorotyrosine. B, inhibition of Csk phosphorylation of
poly(Glu,Tyr) by peptides EDNEYTA (solid column) and
EDNE(FY)TA (hatched column). Enzyme reactions were
carried out with ATP (10 µM) and poly(Glu,Tyr) (10
µg/ml) and the indicated amount of peptide inhibitor. Kinase
activity is shown as relative to control. C, Csk-mediated
phosphorylation of EDNE(F
Y)TA monitored by
F
NMR. Reactions were carried out as described under ``Materials and
Methods.''
F NMR (376 MHz) spectra were taken of the
crude reaction mixtures and shown as minus Csk (a) and plus
Csk (b) (horizontal scale in ppm). D, Lineweaver-Burk
plots of Csk with EDNE(F
Y)TA and EDNE(Y)TA as substrates.
Assays were carried out as described under ``Materials and
Methods,'' and the kinetic constants, determined by non-linear
curve fitting, are shown above.
Initially, EDNE(FY)TA was tested as a
Csk competitive inhibitor with poly(Glu,Tyr) as the tyrosine-containing
substrate. As shown, EDNE(F
Y)TA exhibited similar
inhibitory properties to EDNEYTA (see Fig. 2B). This
similarity was surprising given the potency reported for the
tetrafluorotyrosine-containing peptide toward insulin receptor kinase (K
= 4 µM; K
for the tyrosine peptide = 400
µM)(12) . A careful examination of the reported
data in this paper reveals that the D- and L-tetrafluorotyrosine analog-containing peptides show
very similar inhibitory properties (12) when plotted against
varying lysozyme substrate concentrations. This lack of
stereospecificity, as well as the lack of reported data comparing the
corresponding tyrosine and tetrafluorotyrosine-containing peptides as
inhibitors of lysozyme phosphorylation(12) , led us to
question the K
determinations and mechanistic
interpretations.
The peptide EDNE(FY)TA was next
investigated as a Csk substrate. As mentioned, the
tetrafluorotyrosine-containing peptide was reported not to be a
substrate for insulin receptor tyrosine kinase (12) . Much to
our surprise, EDNE(F
Y)TA was found to be a substrate for
Csk by monitoring ADP formation using a spectrophotometric coupled
assay(15) . A large scale reaction with EDNE(F
Y)TA
was followed by
F NMR. As shown in Fig. 2C, there is nearly complete conversion of
unphosphorylated EDNE(F
Y)TA to phosphorylated
EDNE(F
Y)TA catalyzed by Csk as evidenced by the downfield
shifting of the three fluorine signals. Note that the signals at
-141 and -160 ppm of EDNE(F
Y)TA represent the
fluorines closest to the hydroxyl because their chemical shifts are
moved most downfield by phosphorylation.
Remarkably,
EDNE(FY)TA was a very efficient Csk substrate, with a K
(12 ± 1 mM) and k
(18 ± 0.9 min
) quite
similar to the tyrosine-containing peptide (Fig. 2D).
This result is particularly striking in light of the reported results
with the tetrafluorotyrosine-containing gastrin peptide and the insulin
receptor tyrosine kinase(12) . Although the lack of the
tetrafluorotyrosine-containing gastrin peptide to act as a tyrosine
kinase substrate may have been due to the fact that a different
(insulin receptor) tyrosine kinase was being studied, several other
possible explanations exist. One is the instability of the
phosphorylated product in their assay (which involved 5 washes with the
strong acid phosphoric acid). A second is that the equilibrium constant
for the reaction may lie far to the left (favoring ATP). Note that the
free energy of hydrolysis of the
adenylyl-O-tyrosine-phosphodiester bond was measured to be
similar (within 1 kcal/mol) to the free energy of hydrolysis of the
pyrophosphate bond of ATP(20) . In our case with
EDNE(F
Y)TA, the reaction is driven toward phosphorylation
by coupling to pyruvate kinase and lactate dehydrogenase.
The
implications of EDNE(FY)TA serving as an efficient Csk
substrate (and not a potent inhibitor) significantly altered our
perception of the catalytic mechanism of Csk. It is assumed that the
chemical step in EDNE(Y/F
Y)TA phosphorylation reactions is
at least partially rate-determining since their k
values are 2-3-fold lower than for Csk-mediated
poly(Glu,Tyr) phosphorylation(10) . If stoichiometric
deprotonation of the tyrosine phenol prior to ATP attack were
occurring, one would expect that the tyrosine-containing substrate
would be significantly more reactive as a nucleophile compared with the
trifluorotyrosine-containing substrate. In this case, the
nucleophilicity of the trifluorophenoxide anion versus the
unsubstituted phenoxide anion could differ by as much as
10
. Alternatively, enzymatic phosphoryl transfer could
follow a dissociative mechanism, whereby ADP-phosphoryl covalent bond
breaking is slow (forming a metaphosphate) and precedes nucleophilic
attack. A dissociative mechanism, while not inconceivable, is
inconsistent with the thio effect observed with ATP
S and the
wild-type Csk reaction (see below). It would also be contrary to the
generally accepted view about how kinase enzymes work(21) .
Furthermore, if Csk obeyed a dissociative mechanism, early tyrosine
deprotonation by a catalytic base would serve no useful role in
facilitating phosphoryl transfer.
The most plausible interpretation
of the trifluorotyrosine/Csk experiments is that deprotonation of the
natural tyrosine substrate occurs principally after Tyr O-P bond
formation in the enzymatic reaction. In this way, the nucleophilicity
of the trifluorophenoxide anion could be expected to be comparable with
the largely protonated phenol. ()
As the above studies led us to question the importance of catalytic base function in the Csk kinase reaction, the site-directed Csk mutant D314E was generated. Aspartate 314 is one of 4 invariant protein kinase amino acid residues(22) . In protein kinase A, the corresponding aspartate (Asp-166) has been identified near the substrate serine hydroxyl and predicted to be the catalytic base for this enzyme from x-ray crystallographic analysis(23) , although additional roles for this residue have been considered(3) . Note that although Csk DNA constructs for D314N and D314A were also generated, protein expression was not detectable with these mutants.
The Csk mutant
D314E had greatly reduced enzyme activity vs. the wild-type enzyme. K and k
values
were measured for D314E Csk. The K
values for ATP
(35 ± 8 µM) and poly(Glu,Tyr) (88 ± 12
µg/ml) are rather similar to those of wild-type Csk (ATP K
= 12 ± 1 µM;
poly(Glu,Tyr) K
= 48 ± 2 µg/ml),
but the k
is reduced by a factor of
approximately 10
(k
for wild-type
Csk
40 min
; k
for D314E
Csk
0.005 min
)(10) . That this enzyme
activity in D314E Csk was not due to trace wild-type contamination was
most convincingly demonstrated by examining the sucrose microviscosity
effect. Behaving similar to the microviscosity effect on the ATP
S,
wild-type Csk reaction (slope = -0.22 ±
0.03)(10) , there is a modest fall in k
-control/k
-viscogen with
increasing levels of sucrose for the ATP, D314E Csk reaction (slope
= -0.28 ± 0.06) (see Fig. 3). This is
clearly different from the sucrose microviscosity effect on the ATP Csk
reaction (slope = +0.42 ± 0.04) (see Fig. 3).
It further suggests that as expected the chemical step is fully
rate-determining in both the Csk mutant D314E, ATP reaction, and the
ATP
S, wild-type Csk reaction.
Figure 3:
Viscosity studies with D314E Csk. k-control/k
-viscogen as a
function of increasing microviscosity from sucrose for wild-type (WT) and D314E Csk. Data and plots of wild-type Csk are taken
from (10) . Apparent k
values for D314E
were measured with fixed and near saturating (
3 K
) concentrations of poly(Glu,Tyr) (500
µg/ml) and ATP (100 µM). K
values measured at the extremes of relative viscosity (1 and
3.1) for poly(Glu,Tyr) and ATP were minimally (less than a factor of 2)
affected by viscosity.
The substantial rate loss
associated with D314E Csk compared with wild-type Csk was very
surprising given the trifluorotyrosine results described above. That
large structural perturbations in D314E Csk could account for the
larger than anticipated k effect cannot be ruled
out. The rather minor K
changes for both ATP and
poly(Glu,Tyr) with D314E Csk as well as a similar sucrose
microviscosity effect to the wild-type Csk ATP
S reaction weaken
such an argument, however. Further support for the structural integrity
of D314E Csk comes from data using phospho-Ultrogel affinity
chromatography (11) and native polyacrylamide gel
electrophoresis, where behavior proved to be identical to wild-type Csk
(data not shown).
As the trifluorotyrosine studies argued against
early tyrosine deprotonation in the Csk catalytic mechanism, it was not
surprising that, like the apparent k of the wild
type Csk reaction, k
is unaffected by increasing
the pH of the D314E Csk kinase reaction from 7.4 to 8.8. Furthermore,
the deuterium solvent kinetic isotope effect on k
measured with D314E Csk (at pH 7.4) is actually modestly inverse (k
H/k
D = 0.55
± 0.08), arguing against a proton transfer step becoming
dominant in the transition state of the mutant. A significant, standard
kinetic isotope effect (k
H/k
D > 2) might have
been expected with disruption of catalytic base function.
At this
point, we were unable to fully explain the rather dramatic
10-fold rate reduction in the D314E Csk mutant. Clearly,
aspartate 314 could be important in hydrogen bonding and orienting the
tyrosine hydroxyl toward the
-phosphate. Results of the D314E Csk
mutant with ATP
S suggested another important function, however.
The thio effect (k
-ATP/k
-ATP
S) (24) in the wild-type reaction is 16.9 ± 0.6 (corrected
= 34 assuming that the rate of ADP release is equal to the
chemical step)(10) . In contrast, the thio effect in the mutant
reaction is 4.8 ± 0.6. Recently, it has been pointed out that
the thio effect for non-enzymatic (non-acid catalyzed) phosphoryl
transfers is different for phosphotriesters versus phosphodiesters(24) . For phosphotriester reactions, the
usual thio effect is in the range of 10-200(25) , whereas
for phosphodiester reactions, the range is 4-11 (26) .
While enzymatic thio effects can be more complex to interpret because
of subtle changes in substrate-active site interactions, it is tempting
to rationalize the difference in thio effects for wild-type and mutant
Csk as being related to a change in transition state from triester-like
to diester-like.
Triesters typically react much faster
(10-10
-fold) under non-acidic conditions
with nucleophiles than diesters, probably because of the enhanced
electrophilicity of the phosphorus in the
former(27, 28) . Thus one might anticipate a large
rate reduction to be associated with rendering the
-phosphate of
ATP more diester-like in the enzyme active site. For example, one
hydrogen bond donor to one of the oxyanions on the
-phosphorus
could be disrupted in Csk D314E. Extending this proposal, Asp-314 could
actually serve as this hydrogen bond donor in the wild-type enzyme, but
because of its altered side, chain, Glu-314 is unable to fulfill this
role. An alternative possibility is that the wild-type aspartate 314
enforces a structure that allows another active site functional group
to hydrogen bond to the
-phosphate. In this scenario, D314E
mutation indirectly destabilizes a hydrogen bond to the
-phosphate
by altering the active site organization.
Although a
three-dimensional structure of a catalytically active tyrosine kinase
with bound substrates or inhibitors remains to be
reported(29) , it seems reasonable to suggest that the
``catalytic base'' in Csk and other tyrosine kinases may have
a dual role. The ``catalytic base'' in Csk may still be
important in hydrogen bonding the tyrosine phenol and orienting it in
the active site(3) . Concomitantly it could serve to render the
-phosphate more reactive by hydrogen bonding either directly (X = H+) or indirectly (X = metal,
amino acid side chain) (see Fig. 4). ATP activation may be
particularly important in tyrosine kinase reactions where the
nucleophilicity of the attacking oxygen may be reduced by aryl
conjugation compared with alkyl alcohols of serine/threonine kinases.
Figure 4: Proposal for a dual role of Asp-314 in Csk catalysis.