Kinetic analysis of a protein tyrosine kinase reaction transition state in the forward and reverse directions

Article abstract of DOI:10.1021/ja9808393

Protein tyrosine kinases catalyze the transfer of the γ-phosphoryl group from ATP to tyrosine residues in proteins and are important enzymes in cell signal transduction. We have investigated the catalytic phosphoryl transfer transition state of a protein tyrosine kinase reaction catalyzed by Csk by analyzing a series of fluorotyrosine-containing peptide substrates. It was established for five such fluorotyrosine-containing peptide substrates that there is good agreement between the tyrosine analogue phenol pK(a) and the ionizable group responsible for the basic limb of a pH rate profile analysis. This indicates that the substrate tyrosine phenol must be neutral to be enzymatically active. Taken together with previous data indicating a small β(nucleophile) coefficient (0-0.1), these results strongly support a dissociative transition state for phosphoryl transfer. In addition, the β(leaving group) coefficient was measured for the reverse protein tyrosine kinase reaction and shown to be -0.3. This value is in good agreement with a previously reported nonenzymatic model phosphoryl transfer reaction carried out under acidic conditions (pH 4) and is most readily explained by a transition state with significant proton transfer to the departing phenol.

Full text of DOI:10.1021/ja9808393

VOLUME 120, NUMBER 28
J ULY 22, 1998
© Copyright 1998 by the
American Chemical Society
Kinetic Analysis of a Protein Tyrosine Kinase Reaction Transition
State in the Forward and Reverse Directions
Kyonghee Kim and Philip A. Cole*
Contribution from the Laboratory of Bioorganic Chemistry, Rockefeller UniVersity,
New York, New York 10021
ReceiVed March 13, 1998
Abstract: Protein tyrosine kinases catalyze the transfer of the γ-phosphoryl group from ATP to tyrosine residues
in proteins and are important enzymes in cell signal transduction. We have investigated the catalytic phosphoryl
transfer transition state of a protein tyrosine kinase reaction catalyzed by Csk by analyzing a series of
fluorotyrosine-containing peptide substrates. It was established for five such fluorotyrosine-containing peptide
substrates that there is good agreement between the tyrosine analogue phenol pKa and the ionizable group
responsible for the basic limb of a pH rate profile analysis. This indicates that the substrate tyrosine phenol
must be neutral to be enzymatically active. Taken together with previous data indicating a small ânucleophile
coefficient (0-0.1), these results strongly support a dissociative transition state for phosphoryl transfer. In
addition, the âleaving group coefficient was measured for the reverse protein tyrosine kinase reaction and shown
to be -0.3. This value is in good agreement with a previously reported nonenzymatic model phosphoryl
transfer reaction carried out under acidic conditions (pH 4) and is most readily explained by a transition state
with significant proton transfer to the departing phenol.
Protein tyrosine kinases catalyze the transfer of the γ-phos-
phoryl group of ATP to proteins on tyrosine residues (Figure
1). These enzymes are critical for cell signal transduction in a
variety of physiologic contexts.1 Because of their importance
in embryonic development, cancer, neurobiology, endocrinology,
and immunology, protein tyrosine kinases have been intensively
Figure 1. Protein tyrosine kinase-catalyzed reaction.
* To whom correspondence should be addressed: tel, (212) 327-7241;
studied in recent years.2 There is a major pharmaceutical effort
fax, (212) 327-7243; e-mail, cole@rockvax.rockefeller.edu.
(1) (a) Hunter, T. Cell 1995, 80, 225-236. (b) Shokat, K. M. Chem.
Biol. 1995, 2, 509-514.
to develop specific inhibitors of these enzymes as drugs.3
(2) (a) Cheng, H. J.; Flanagan, J. G. Cell 1994, 79, 157-168. (b) Banker,
N.; Evers, B. M.; Hellmich, M. R.; Townsend, C. M. Surg. Oncol. 1996, 5,
201-210. (c) Creedon, D. J.; Tansey, M. G.; Baloh, R. H.; Osborne, P. A.;
Lampe, P. A.; Fahrner, T. J.; Heuckeroth, R. O.; Millbrandt, J.; Johnson,
E. M. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 7018-7023. (d) White, M.
F.; Kahn, C. R. J. Biol. Chem. 1994, 269, 1-4. (e) Heyeck, S. D.; Wilcox,
H. M.; Bunnell, S. C.; Berg, L. J. J. Biol. Chem. 1997, 272, 25401-25408.
(3) (a) Levitzki, A.; Gazit, A. Science 1995, 267, 1782-1788. (b)
Mohammadi, M.; McMahon, G.; Sun, L.; Tang, C.; Hirth, P.; Yeh, B. K.;
Hubbard, S. R.; Schlessinger, J. Science 1997, 276, 955-960.
The amino acid sequences and three-dimensional structures
of protein serine/threonine-specific kinases and tyrosine-specific
kinases are highly conserved.4 Many of the amino acids that
(4) (a) Madhusudan; Trafny, E. A.; Xuong, N.-H.; Adams, J. A.; Ten
Eyck, L. F.; Taylor, S. S.; Sowadski, J. M. Protein Sci. 1994, 3, 176-187.
(b) Johnson, L. N.; Noble, M. E. M.; Owen, D. J. Cell 1996, 85, 149-158.
(c) Xu, W.; Harrison, S. C.; Eck, M. J. Nature 1997, 385, 595-601. (d)
Sicheri, F.; Moarefi, I.; Kuriyan, J. Nature 1997, 385, 602-608.
S0002-7863(98)00839-7 CCC: $15.00 © 1998 American Chemical Society
Published on Web 07/03/1998

6852 J. Am. Chem. Soc., Vol. 120, No. 28, 1998
Kim and Cole
Figure 2. Associative vs dissociative transition states for phosphoryl transfer. ROH is the nucleophile (tyrosine phenol in this work) attacking the
γ-phosphoryl group of ATP, and ADP is the leaving group. Associative transition state (path A) in this work is defined as more than 50% bond
formation between the nucleophilic oxygen and the phosphorus, which occurs with at least 50% leaving group residual bond formation present.5
Dissociative transition state (path B) in this work is defined as less than 50% bond formation between the nucleophile and the phosphorus, which
occurs before the leaving group-phosphorus bond is at least 50% broken.
make up the catalytic active site of protein kinases are identical
within the superfamily. The nature of the transition state in
protein kinase reactions, which fundamentally sets the bound-
aries for the biologically topical issues of regulation and
substrate selection, has not yet been fully elucidated.
for a wide variety of nonenzymatic phosphate monoester
phosphoryl transfer reactions and has been shown to be small
(0-0.3) for this class of reactions.10 A small ânuc is expected
for a dissociative mechanism since there is little bond formation
between the nucleophile and phosphorus in the transition state.
In contrast, nonenzymatic phosphate triester reactions typically
exhibit large ânuc values (0.5-0.9) more consistent with an
associative mechanism.7
Linear free-energy relationships are often more difficult to
obtain in enzyme systems because of the typically strict
requirements for substrate active site binding. Thus, obtaining
a series of homologous substrates that differ in nucleophile pKa
but bind to the enzyme active site with similar affinity and
orientation is often not possible. In principle, a protein tyrosine
kinase reaction is amenable to such studies because the substrate
tyrosine aromatic ring can be substituted in ways that might
not be too disruptive to binding or catalysis. Recently, using a
series of fluorotyrosine-containing peptides as substrates, a ânuc
for kcat and kcat/Km of 0-0.1 was reported for protein tyrosine
kinase Csk-catalyzed phosphorylation.11 By comparing the
reaction rates at pH 7.4 and pH 6.6 for several of the analogues,
it was suggested that the neutral phenol rather than the
phenoxide anion was the preferred nucleophile for the enzyme
reaction.11
Measurements of the âleaving group can provide complementary
information about the transition states of phosphoryl transfer
reactions. Large negative âleaving groups (ca. -1) are usually
observed with nonenzymatic phosphate monoester phosphoryl
transfer reactions at neutral pH, indicating that the transition
state shows significant negative charge buildup on the leaving
group and therefore a high degree of bond cleavage between
phosphorus and the leaving group. Under acidic conditions
(e.g., pH 4), a more modest âleaving group value (-0.3) is
associated with hydrolyses of phosphate monoesters because
proton transfer to the departing oxygen atom is a critical feature
of the transition state.12
In general, the mechanisms of nonenzymatic phosphoryl
transfer reactions are fairly well-understood.5 Nonenzymatic
phosphate monoester reactions follow dissociative mechanisms
where the bond between phosphorus and the leaving group is
largely broken in the transition state and the bond between the
nucleophile and phosphorus is minimally formed in the transition
state (Figure 2). It has been argued that enzymes can render
phosphoryl transfer transition states more associative by sur-
rounding the attacked phosphate with positively charged groups,
making the phosphate a better electrophile by neutralizing its
negative charge.6 Justification for this analysis has in part been
based on nonenzymatic phosphate triester reactions that show
associative character.7 A judiciously positioned active site base
revealed by crystallographic studies of protein kinases has been
suggested to make the hydroxyl nucleophile more reactive in
enzymatic reactions, facilitating an associative phosphoryl
transfer reaction mechanism for protein kinases.8 However, a
long reaction coordinate distance of 5.2 Å between the entering
hydroxy nucleophile and the ATP γ-phosphoryl group in the
serine/threonine protein kinase A active site has been taken as
evidence for a dissociative mechanism.6c
Linear free-energy relationships have proven useful in clarify-
ing reaction mechanisms in physical organic chemistry.9 The
Brønsted nucleophile coefficient (ânuc), a measure of the role
of the nucleophile in the transition state, has been determined
(5) (a) Admiraal, S. J.; Herschlag, D. Chem. Biol. 1995, 2, 729-739.
(b) Maegley, K. A.; Admiraal, S. J.; Herschlag, D. Proc. Natl. Acad. Sci.
U.S.A. 1996, 93, 8160-8166.
(6) (a) Mildvan, A. S.; Fry, D. C. AdV. Enzymol. Relat. Areas Mol. Biol.
1987, 59, 241-313. (b) Schlichting, I.; Reinstein, J. Biochemistry 1997,
36, 9290-9296. (c) Granot, J.; Mildvan, A. S.; Bramson, H. N.; Kaiser, E.
T. Biochemistry 1980, 19, 3537-3543.
In an effort to improve understanding of protein kinase
catalysis, in this study we report the results of a detailed kinetic
(7) (a) Khan, S. A.; Kirby, A. J. J. Chem. Soc. B, 1970, 1172-1182. (b)
Epstein, J.; Plapinger, R. E.; Michel, H. O.; Cable, J. R.; Stephani, R. A.;
Billington, C. A.; West, G. R. J. Am. Chem. Soc. 1964, 86, 3075-3084.
(8) (a) Bossemeyer, D.; Engh, R. A.; Kinzel, V.; Ponstingl, H.; Huber,
R. EMBO J. 1993, 12, 849-859. (b) Zheng, J.; Knighton, D. R.; Ten Eyck,
L. F.; Karlsson, R.; Xuong, N.-H.; Taylor, S. S.; Sowadski, J. M.
Biochemistry 1993, 32, 2154-2161.
(10) (a) Kirby, A. J.; Varvoglis, A. G. J. Chem. Soc. B 1968, 135-141.
(b) Herschlag, D.; Jencks, W. P. J. Am. Chem. Soc. 1987, 109, 4665-
4674.
(11) Kim, K.; Cole, P. A. J. Am. Chem. Soc. 1997, 119, 11096-11097.
(12) Kirby, A. J.; Varvoglis, A. G. J. Am. Chem. Soc. 1967, 89, 415-
423.
(9) Jencks, W. P. Catalysis in Chemistry and Enzymology; McGraw-
Hill: New York, 1969.

Tyrosine Kinase Reaction Transition State
J. Am. Chem. Soc., Vol. 120, No. 28, 1998 6853
complex when plotting log(kcat) vs pH.15a Four of the fluoro-
tyrosine peptides and the unfluorinated tyrosine-containing
peptide were assayed (kcat/Km measurements) with Csk covering
the pH range 6-8.6 (Figure 4). It proved impossible to obtain
complete data sets for the corresponding kcat plots because the
Km values in some cases were too high to be accurately
measured.
A plot of the log kcat/Km vs pH for the unfluorinated tyrosine-
containing peptide showed an increase in rate from pH 6 to pH
7 and then reached a plateau (Figure 4A). This behavior fits
reasonably well to a model with a single ionizable group
(corresponding to pK1) with pKa near 7 that facilitates phos-
phoryl transfer in its deprotonated form. Given this pKa value,
it is unlikely that the proposed ionizable group comes from the
peptide substrate since in free solution EDNEYTA lacks such
a pKa. At this time, it is unclear whether this proposed ionizable
group is involved in substrate binding or phosphoryl transfer,
although the former is favored because of the lack of a pH rate
effect in this region for Csk-catalyzed phosphorylation of the
substrate poly(glu,tyr).
Kinase kinetic analysis of the substrate fluorotyrosine peptide
derivatives showed evidence of a similar titratable group (pK1)
to that found with the unfluorinated peptide, although with a
slightly higher pKa (7.5-8) (Figure 4B-E). However, instead
of a plateau at the higher pH, the kcat/Km decreased above pH
8 in all cases, suggestive of an additional group (pK2) leading
to decreased kinase activity in its deprotonated form. The
theoretical fit of the data in Figure 4B-E revealed that the value
of pK2 was lower than the value of pK1 for these substrates.
The pKa values of the fluorotyrosine phenols measured spec-
trophotometrically agree nicely with those extrapolated from
the kinetic analyses (pK2) in plots of log(kcat/Km) vs pH (Table
1). These results provide convincing evidence that the neutral
phenol of the tyrosine and fluorotyrosine derivatives rather than
the phenoxide anion is the reactive nucleophile in the enzymatic
reaction. The more chemically reactive phenoxide anion species
in contrast appears inactive in the enzymatic phosphoryl transfer
reactions.
Deprotonation of the tyrosine hydroxyl is a necessary feature
of phosphoryl transfer to the phenol along the reaction coor-
dinate. Therefore, it is important to consider the circumstances
that could account for the neutral phenol behaving as a ‘more
reactive’ nucleophile than the free phenoxide anion in the
enzyme reaction. In an associative mechanism, with significant
participation of the nucleophile in the transition state, a typical
ânuc value is 0.6.7 Since the pKa values of the phenol and
phenoxide anions differ by ∼17 units,15b the phenoxide anion
is expected to react approximately 1010-fold faster than the
neutral phenol assuming identical orientation and binding
behavior. The transition state energy barrier for phenoxide ion
nucleophilic attack would thus be approximately 14 kcal/mol
lower as compared to the neutral phenol. In order for the
phenoxide ion to be a less successful nucleophile in the
associative mechanism (if the ânuc ) 0.6), disrupted binding
forces costing more than 14 kcal/mol energy would apparently
be required. While the loss of a hydrogen bond and/or
electrostatic repulsion in the neutral versus anionic species could
contribute in part to such a binding disruption, it would be
difficult to account for such a large energy change by these
factors alone.
Figure 3. Fluorotyrosine analogues used in this work.
investigation on protein tyrosine kinase-catalyzed phosphoryl
transfer reactions to and from fluorotyrosine peptide substrates.
These studies further support the proposal of a dissociative
reaction mechanism with the neutral phenol acting as the
preferred substrate nucleophile.
Results and Discussion
Synthesis of Fluorotyrosine Amino Acids and Peptides.
Standard total chemical synthesis of the nine fluorotyrosine
amino acids (Figure 3) used in these investigations would be
laborious. For example, the known amino acid tetrafluoroty-
rosine (10) is obtained from hexafluorobenzene in five steps to
afford racemic amino acid.13 Syntheses of several of the other
di- and trifluorotyrosines could be expected to be at least as
difficult by standard routes. Separation and characterization of
the individual enantiomers is also a tedious process. Fortunately,
the enzyme tyrosine phenol-lyase has been shown to be a highly
efficient and rather general catalyst for the incorporation of
substituted phenols into L-tyrosine derivatives.14 Gram quanti-
ties of the fluorotyrosine amino acids using the recombinant
enzyme tyrosine phenol-lyase were prepared from the phenols
in the presence of pyruvate and NH3. To generate tetrafluo-
rotyrosine (10), extended reaction times and greater quantities
of tyrosine phenol-lyase enzyme were required. The reduced
processing efficiency of tetrafluorophenol may be due to a
combination of increased steric bulk and/or a decreased ability
to stabilize positive charge in the aryl group in the transition
state.
The unprotected fluorotyrosine amino acids (X, Figure 3)
were reacted with Fmoc-O-succinimidyl carbonate and the Fmoc
amino acids were used to generate the peptides NH2-Glu-Asp-
Asn-Glu-X-Thr-Ala-CO2H by automated solid-phase peptide
synthesis using a 0.1-0.25 mmol scale. Peptides were obtained
in approximately 50% yield after purification.
Phosphorylation vs pH. Given the preliminary pH rate
studies presented for the various fluorotyrosine peptides reported
earlier,11 it was important to try to fully establish the true
correlation between the pKa of the phenol and the kinetics of
the kinase reaction in a more extensive analysis. In principle,
the titration of acid/base groups versus pH should reflect the
free substrate and enzyme form when analyzing log(kcat/Km) vs
pH whereas they should reflect the bound enzyme/substrate
(13) Filler, R.; Ayyangar, N. R.; Gustowski, W.; Kang, H. H. J. Org.
Chem. 1969, 34, 534-538.
(14) (a) Hebel, D.; Furlano, D. C.; Phillips, R. S.; Koushik, S.; Creveling,
C. R.; Kirk, K. L. Bioorg. Med. Chem. Lett. 1992, 2, 41-44. (b) Nagasawa,
T.; Utagawa, T.; Goto, J.; Kim C.-J.; Tani, Y.; Kumagai, H.; Yamada, H.
Eur. J. Biochem. 1981, 117, 33-40.
However, in a dissociative mechanism where a typical value
of ânuc is 0.1, the phenoxide anion would only be expected to
(15) (a) Dixon, M.; Webb, E. C. Enzymes; Academic Press: New York,
1964. (b) Wu, C. Y.; Arnett, E. M. J. Am. Chem. Soc. 1960, 82, 5660-
5665.

6854 J. Am. Chem. Soc., Vol. 120, No. 28, 1998
Kim and Cole
B
A
C
D
E
Figure 4. Profiles of log(kcat/Km) vs pH for peptides containing fluorotyrosine analogues 1, 6, and 8-10 (A-E, respectively). Symbols: Diamonds,
Bis-Tris-HCl buffer; triangles, Mops-Na buffer; circles, Tris-HCl buffer. See Experimental Section for other details. In this work, the pKa corresponding
to the acidic limb of the profiles is defined as pK1, and that corresponding to the basic limb is defined as pK2. The calculated acidity constants (
standard error for each of the plots are as follows: A, pK1 ) 7.0 ( 0.1; B, pK1 ) 7.9 ( 0.1, pK2 ) 7.3 ( 0.1; C, pK1 ) 7.6 ( 0.2; pK2 ) 6.9 (
0.3; D, pK1 ) 7.7 ( 0.3; pK2 ) 6.3 ( 0.2; E, pK1 ) 7.8 ( 0.8; pK2 ) 5.6 ( 0.8.
react about 50-fold faster as compared to the neutral phenol,
corresponding to approximately 2 kcal/mol lowering of the
transition state energy barrier. Under these conditions, the loss
of a hydrogen bond and/or electrostatic repulsion could dominate
the expected rate effects due to loss of nucleophilicity. Thus
the combination of a low ânuc and a requirement for a neutral
phenol are persuasive evidence for a dissociative mechanism.
Table 1. Substrate Phenolic Spectrophotometric pKa Values
Correlated with Enzyme (Forward Reaction) Kinetic pKa Valuesa
tyrosine analog
spectrophotometric pKa
kinetic pKa
1
6
8
9
10
10
>8
7.6
6.6
6.1
5.2
7.3 ( 0.1
6.9 ( 0.3
6.3 ( 0.2
5.6 ( 0.8
a The kinetic pKa values are defined as the pKa values (the basic
limbs) from Figure 4. For details of the measurements, see the
Experimental Section.
What specific factors could be responsible for the lack of
reaction of the negatively charged nucleophile in the Csk kinase

Tyrosine Kinase Reaction Transition State
J. Am. Chem. Soc., Vol. 120, No. 28, 1998 6855
reaction?16 Possibilities include repulsion by the negatively
charged phosphate oxygens17 and/or the highly conserved active
site aspartate carboxylate anion (asp-314 in Csk).4 There are
several reports discussing the potential repulsion of negatively
charged nucleophiles by the phosphate oxygens in phosphate
monoesters.17,18 Approximately 2 kcal/mol electrostatic repul-
sion energy between anionic nucleophiles and phosphate mo-
noesters has been estimated from model systems.17 In combi-
nation with the predicted loss of hydrogen bonding and gain of
electrostatic repulsion between the asp-314 carboxylate and the
phenoxide anion, it is reasonable to rationalize the 4-5 kcal/
mol energy loss required to explain the decreased efficiency of
the phenoxide anion as nucleophile in the enzymatic reaction.
Analysis of the kcat vs pH for enzymes can provide informa-
tion about the pKa values of groups in the bound complex.
Because of the already low affinity of the peptide substrates
used in this work for Csk, accurate kcat values could not be
obtained for the fluorotyrosine analogues at some of the pHs.
However, from the available data (not shown), it could be
estimated that the kcat values vs pH showed similar behavior to
the kcat/Km vs pH. The pKa of the substrate tyrosine is probably
(2 units in the enzyme active sites compared to its value in
free solution. Theoretically, altering the pKa of the nucleophile
would serve little purpose in facilitating a dissociative mech-
anism.
Figure 5. HPLC trace of phosphopeptide and peptide containing
fluorotyrosine derivative (8). HPLC conditions were as follows: C-18
analytical column, water:acetonitrile gradient (0.05% trifluoroacetic
acid); 0-10 min, 0-10% acetonitrile; 10-20 min, 10-15% acetoni-
trile; 20-30 min, 15-25% acetonitrile. UV monitoring at 214 nm was
carried out. Peak A represents phosphopeptide (retention time, 16 min)
and peak B corresponds to unphosphorylated peptide (retention time,
24 min).
standard hexokinase/glucose-6-phosphate dehydrogenase spec-
trophotometric assay20 due to high background. Instead, an
HPLC assay with direct monitoring of conversion to the
dephosphorylated peptide was employed. Background hydroly-
sis rates of the phosphopeptides under these conditions (pre-
sumed to be related to trace quantities of contaminating
phosphatases) were measured by omitting ADP from the
reaction mixtures and for six of the peptides shown to be less
than 15% of the total rates. In the four cases where the
background rate was significant (40-70% of the total rate, see
Experimental Section), it was found to be reproducible and was
subtracted from the total rate to obtain the true reverse kinase
rates. Product formation was shown to be linear with time for
at least 1 h and with enzyme concentration in the range of 1-10
µM. Enzyme activity versus phosphopeptide concentration was
found to be linear in the 1-4 mM range for all peptides
examined so that kcat/Km (but not kcat) could be readily measured
under these conditions. Studies in the forward kinase direction
and previous inhibitor analyses indicate that the fluorotyrosines
bind with similar affinity (over a 3-fold range)11,16 so the absence
of direct Km measurements for the reverse reaction was not
considered a liability. The rate (kcat/Km) for the standard tyrosine
(1)-containing peptide allows an estimate of the equilibrium
constant ([phosphopeptide][ADP]/[peptide][ATP]) of approxi-
mately 23 (at pH 7.4, in the forward kinase direction) assuming
the ADP Km is approximately 5-fold greater than the Km for
ATP and the mechanism is ‘rapid equilibrium random’ with
Mg.21 This is in fairly good agreement with the value of 10
measured with a different peptide substrate with the Src
enzyme.19
âleaving group for the Reverse Reaction. In principle, obtaining
the âleaving group for a protein tyrosine kinase reaction would
involve studying ATP analogues with a range of pKa values
for the â-phosphate of the leaving ADP moiety. It is unlikely
that the necessary modifications would be compatible with
enzymatic catalysis because they would significantly alter the
steric and electronic properties of the ADP molecule. In
contrast, a more realistic goal appeared to be evaluating the
âleaving group for the reverse kinase (ATP synthesis) reaction. The
equilibrium constant was expected to be approximately 10 for
tyrosine phosphorylation in the forward direction at neutral pH.19
Kinetic analysis of the reverse reaction with ADP as the
nucleophile and the tyrosine phenol as the leaving group
therefore seemed plausible since the requisite substituted tyrosine
derivatives were already accessible.
The necessary phosphopeptides were obtained by enzymatic
phosphorylation of the fluorotyrosine-containing peptides using
recombinant Csk. The phosphorylated peptides were very well
separated from the unphosphorylated forms by reverse-phase
HPLC using a standard water:acetonitrile gradient (see Figure
5), and electrospray ionization mass spectrometry confirmed
their identity. These phosphopeptides were stable in frozen
solution at -80 °C for extended periods.
Csk enzymatic assay of the phosphopeptides in the ATP
synthesis direction was carried out. The reverse kinase reactions
proved to be too slow to be accurately monitored with the
Analysis of the log(kcat/Km) vs leaving group pKa is shown
in Figure 6. The slope which equals the âleaving group was
calculated by linear regression to be -0.33 ( 0.06. This value
is considerably less negative than that commonly observed for
phosphate monoester phosphoryl transfer reactions5a and would
even be low for diesters (typically -0.9 to -1.1)18 and triesters
(typically -0.4 to -1).7a One possibility for this small effect
is that the more highly fluorinated compounds do not bind as
well to the enzyme, which could offset more marked kcat rate
effects. Evidence against this possibility comes from studies
in the forward direction where the Km values (which are likely
(16) The simple possibility that the phenoxide anion is unable to bind
to Csk is unlikely. Titrating the peptide containing tetrafluorotyrosine (1-
16 mM) as an inhibitor (at pH 7.4 in which approximately 99% exists in
the phenoxide anionic form) of Csk phosphorylation [fixed substrate
concentration of EDNEYTA (4 mM)] showed that it was a linear inhibitor
with a Ki of 4.6 ( 0.5 mM assuming a competitive inhibition model. This
Ki is in good agreement with the Km of EDNEXTA peptides11 at this pH as
well as with the Ki of EDNEFTA (demonstrated to be a linear competitive
inhibitor versus poly(glu, tyr) with a Ki of 4.9 ( 0.7 mM21 and the deduced
Ki values of EDNEYTA and EDNEXTA (X ) 9) versus the substrate poly-
(glu, tyr).24
(17) Herschlag, D.; Jencks, W. P. J. Am. Chem. Soc. 1989, 111, 7587-
7596.
(18) Kirby, A. J.; Younas, M. J. Chem. Soc. B. 1970, 1165-1172.
(19) Boerner, R. J.; Barker, S. C.; Knight, W. B. Biochemistry 1995,
34, 16419-16423.
(20) Oliver, I. T. J. Biol. Chem. 1955, 61, 116-122.
(21) Grace, M. R.; Walsh, C. T.; Cole, P. A. Biochemistry 1997, 36,
1874-1881.

6856 J. Am. Chem. Soc., Vol. 120, No. 28, 1998
Kim and Cole
Dissociative Mechanism for Protein Kinases. Through the
use of ânuc measurements, pH rate analyses, and âleaving group
studies, a picture emerges of the protein kinase mechanism in
which the transition state of the reaction is dissociative. In such
a mechanism, there is relatively little bond formation between
the attacking nucleophile (serine or tyrosine) and the phosphorus
prior to departure of the leaving group (ADP). Although it is
very hard to prove enzymatic mechanisms with absolute
certainty, there is no existing evidence that contradicts this
model. Short reaction coordinate distances between the entering
serine oxygen and the γ-phosphorus of enzyme-bound ATP of
2.7-3.1 Å were obtained by X-ray crystallography studies on
protein kinase A4a,8 while a longer reaction coordinate distance
(5.2 Å) was obtained by NMR.6c A recent X-ray crystal-
lographic analysis of insulin receptor tyrosine kinase showed a
5 Å reaction coordinate distance in a ternary complex.23 All
of these studies have required the use of unreactive substrate
analogues that make their reliability uncertain. Although a large
kcat thio effect for a Csk reaction was initially taken as evidence
of an associative mechanism,24 more detailed studies with
various thiophilic metal ions have argued that the thio effect is
largely a matter of impaired metal coordination rather than
electronegativity changes due to sulfur.21 Furthermore, 18O-
isotope effects, while small and sometimes difficult to interpret,
have generally supported the concept that other phosphate
monoester enzymatic reactions are dissociative.27
From a theoretical standpoint, a cogent argument has been
advanced that if the enzyme were to render the transition state
more associative than the transition state of the nonenzymatic
reaction, it would have to supply greater stabilization than to
stabilize a dissociative mechanism.5 Perhaps the most unsettling
aspect of dissociative enzymatic mechanisms for protein kinases
is that it is not so obvious how precise product formation could
be achieved. It is true that some protein kinases have low
ATPase activity, but the presumption is that there must be rigid
geometric constraints for the binding of the nucleophile before
leaving group departure is activated. Many of the active site
residues would have key orientational roles in this model. In
protein kinases, the key activating residues would be those that
effect rupture of the γ-phosphate-â-phosphate bridging oxygen
bond. While there is no hard information on this point, the
possible key participants would be one of the divalent ions and
or a conserved active site lysine.4
Implications of a Dissociative Mechanism. There is a major
effort in the cell signal transduction community in characterizing
protein kinases in order to understand the basis of substrate
selectivity and to discern how the enzymes are regulated.1,2,4
Pharmacologists are interested in designing specific inhibitors
of these enzymes that might have therapeutic value for a wide
range of diseases.3 The predicted dissociative transition state
distance between the entering nucleophilic hydroxyl oxygen and
the departing ADP oxygen is on the order of 1-2 Å larger than
in an associative transition state.28 To model the role of amino
acids in the active site that would stabilize such a transition
state and dictate substrate selection, it is helpful to have
knowledge of the fundamental strategy the enzyme uses in
effecting catalysis. Likewise, in describing the “inactive” versus
“active” structures of the enzymes, a major goal of structural
biologists, it would seem essential to define the electronic
requirements of the “active” state. Successful transition state
analogue inhibitor design would appear to require the develop-
Figure 6. Brønsted plot of the log(kcat/Km) versus the tyrosine phenol
pKa values for the reverse Csk tyrosine kinase reactions with substrates
ADP and phosphopeptides (with tyrosine derivatives 1-10). See the
Experimental Section for details. The âleaving group (slope ( standard error)
was calculated using linear regression and found to be -0.33 ( 0.06.
equal to Kd values) for the various peptides were shown to be
similar (within a 3-fold range) regardless of the number of
fluorines in the peptides. A second possibility is that the
reaction is not limited by chemistry. This possibility seems
unlikely since the reactions in the forward direction (which are
faster) are very likely to be limited by chemistry, and ATP and
peptide likely have fast off rates.11,21 A third possible explana-
tion is that the reverse kinase reaction is highly associative. This
is very unlikely given the law of microscopic reversibility based
on the fact that the forward direction is likely dissociative for
reasons discussed above.
The most likely explanation for the low âleaving group is that
the transition state involves protonation of the leaving group
by an active site acid group, which would attenuate the buildup
of effective negative charge on the departing oxygen.22 Such
an effect has been observed in the hydrolysis of phosphate
monoesters at pH 4. In fact, a nearly identical âleaving group value
of -0.27 was observed in this nonenzymatic reaction model
system.12 The facilitation of leaving group departure would
appear to be a key feature of dissociative transition states, and
the use of an active site acid to protonate the leaving group
oxygen would accomplish this objective. As mentioned above,
the active nucleophilic species in the forward kinase direction
appears to be the neutral phenol. Since the law of microscopic
reversibility requires that the protonation state of the phenol in
the transition state be identical in both directions, a large
negative âleaving group effect (e.g., -1) would be incompatible
with the mechanistic model based on studies of the forward
kinase direction.
While the identity of the active site acid group is not known
with certainty, it seems most reasonable to suggest that in Csk
it is Asp-314. A recent crystal structure of the insulin receptor
protein tyrosine kinase revealed that this conserved aspartate
hydrogen bonds to the nucleophilic hydroxy group.23 Moreover,
mutation of this conserved aspartate in Csk21,24 and the
corresponding residue in protein kinase A25,26 have been shown
to be associated with a reduction in kcat of approximately 104
on the chemical step for both. In the forward direction, the
aspartate is expected to direct and orient the hydroxyl group
toward the γ-phosphate. Its role as a “catalytic base” if any
would be expected to take place well after the tyrosine-phosphate
bond is largely formed.
(22) Williams, A. AdV. Phys. Org. Chem. 1992, 27, 1-55.
(23) Hubbard, S. R. EMBO J. 1997 16, 5572-5581.
(24) Cole, P. A.; Grace, M. R.; Phillips, R. S.; Burn, P.; Walsh, C. T. J.
Biol. Chem. 1995, 270, 22105-22108.
(27) Jones, J. P.; Weiss, P. M.; Cleland, W. W. Biochemistry 1991, 30,
3634-3639.
(25) Gibbs, C. S.; Zoller, M. J. Biochemistry 1991, 30, 8923-8931.
(26) Zhou, J.; Adams, J. A. Biochemistry 1997, 36, 2977-2984.
(28) Mildvan, A. S. Proteins: Struct. Funct. Genet. 1997, 29, 401-
416.

Tyrosine Kinase Reaction Transition State
J. Am. Chem. Soc., Vol. 120, No. 28, 1998 6857
1 H). 19F NMR (D2O); δ -116 (s, 2 F). HRMS calcd. for C9H9-
NO3F2 (MH)+, 218.0629; found, 218.0630.
2,3-Difluorotyrosine (5). 1H NMR (D2O); δ 6.90 (m, 1 H), 6.70
(m, 1 H), 3.95 (dd, J ) 8.0, 5.2 Hz, 1 H), 3.31 (dd, J ) 14.8, 5.2 Hz,
1 H), 2.88 (dd, J ) 14.8, 8.0 Hz, 1 H). 19F NMR (D2O); δ -143 (d,
J ) 18.8 Hz, 1 F), -163 (d, J ) 18.8 Hz, 1 F). HRMS calcd. for
C9H9NO3F2 (MH)+, 218.0629; found, 218.0629.
2,5-Difluorotyrosine (6). 1H NMR (D2O); δ 7.03 (dd, J ) 11.2,
7.6 Hz, 1 H), 6.71 (dd, J ) 11.2, 7.6 Hz, 1 H), 3.95 (dd, J ) 8.0, 5.3
Hz, 1 H), 3.26 (dd, J ) 14.8, 5.3 Hz, 1 H), δ 3.03 (dd, J ) 14.8, 8.0
Hz, 1 H). 19F NMR (D2O); δ -123 (m, 1 F), -143 (m, 1 F). HRMS
calcd. for C9H9NO3F2 (MH)+, 218.0629; found, 218.0627.
3,5-Difluorotyrosine (7). 1H NMR (D2O); δ 6.85 (m, 2 H), 3.93
(dd, J ) 8.4, 4.5 Hz, 1 H), 3.20 (dd, J ) 14.8, 4.5 Hz, 1 H), 2.88 (dd,
J ) 14.8, 8.4 Hz, 1 H). 19F NMR (D2O); δ -135 (brs, 2 F). HRMS
calcd. for C9H9NO3F2 (MH)+, 218.0629; found, 218.0631.
2,3,6-Trifluorotyrosine (8). 1H NMR (D2O); δ 6.24 (m, 1 H), 3.71
(brs, 1 H), 3.07 (d, J ) 14.8 Hz, 1 H), 2.92 (m, 1 H). 19F NMR (D2O);
δ -124 (m, 1 F), -143 (m, 1 F), -169 (m, 1 F). HRMS calcd. for
C9H8NO3F3 (MH)+, 236.0535; found, 236.0538.
2,3,5-Trifluorotyrosine (9). 1H NMR (D2O); δ 6.58 (brs, 1 H),
3.70 (brs, 1 H), 3.10 (d, J ) 14.4 Hz, 1 H), 2.85 (t, J ) 7.2 Hz, 1 H).
19F NMR (D2O); δ -151 (m, 1 F), -158 (m, 1 F), -170 (m, 1 F).
HRMS calcd. for C9H8NO3F3 (MH)+, 236.0535; found, 236.0535.
2,3,5,6-Trifluorotyrosine (10). 1H NMR (D2O); δ 3.92 (m, 1 H),
3.27 (m, 1 H), 3.16 (m, 1 H). 19F NMR (D2O); δ -150 (m, 2 F),
-167 (m, 2 F). HRMS calcd. for C9H7NO3F4 (MH)+, 254.0440; found,
254.0441.
Measurements of Fluorotyrosine Analogue pKa Values. The pKa
values of the fluorotyrosine analogues were determined spectrophoto-
metrically by monitoring the bathochromic shift (ca. 270 nm f 290
nm) that occurs upon conversion of the neutral phenol to the phenoxide
anion. Appropriate pH ranges (3-11) for each analogue were obtained
using the following buffers (30 mM): formate, acetate, Mes, Mops,
Tris, Capso, and Caps along with 150 mM NaCl at 30 °C. Data were
fit to the Hill equation, demonstrating a single proton-transfer titration,
and standard errors for calculated pKa values were (0.2 unit. Previous
work has shown that there is good agreement ((0.3 unit) between
phenol pKa values of the free amino acids and peptides.24
ment of a compound that could fill the relatively larger
molecular space occupied by a dissociative transition state.
Experimental Section
Expression and Purification of Tyrosine Phenol-Lyase. The
protocol is based on the method of Phillips and colleagues.29 Single
colonies of the SVS370 Escherichia coli strain carrying the DNA
plasmid pTZTPL were used to innoculate 3 × 1 L of LB medium
containing ampicillin (100 µg/mL), and the cultures were grown at 37
°C for 20 h in a shaker/incubator. Cells were harvested by centrifuga-
tion (30 min, 10000g) affording 20 g, snap frozen, and stored at -80
°C until needed. The thawed cells were suspended in 80 mL of standard
buffer (0.1 M potassium phosphate, pH 7.0, 0.1 mM pyridoxal
phosphate, 1 mM EDTA, and 5 mM â-mercaptoethanol) and lysed by
passage through a French pressure cell (600 psi). The resultant
suspension was centrifuged for 30 min at 25000g at 4° C, and the
recovered supernatant was treated with 2% w/v protamine sulfate (12
mL) by dropwise addition at room temperature. The resultant cloudy
solution was centrifuged for 30 min at 25000g at 4 °C, and the clear
yellow supernatant was brought to 60% saturation with the addition of
solid (NH4)2SO4 at 4 °C. The resultant suspension was centrifuged
for 30 min at 25000g, and the pellet was dissolved in standard buffer
and dialyzed against standard buffer, 25% saturated with (NH4)2SO4
overnight at 4 °C. The protein solution was loaded onto the previously
equilibrated octyl-Sepharose column (11 cm × 2 cm) with the standard
buffer 25% saturated with (NH4)2SO4 at 1 mL/min. After continuing
to elute with standard buffer 25% saturated with (NH4)2SO4, early
fractions (about 150 mL) containing tyrosine phenol-lyase were
combined, precipitated with 75% saturating (NH4)2SO4, resuspended,
and dialyzed against standard buffer to afford 80 mL of tyrosine phenol-
lyase solution at a concentration of 5 mg/mL protein. A 10% SDS-
PAGE stained with Coomassie showed that tyrosine phenol-lyase was
approximately 70% pure. Lyase activity assay (lactate dehydrogenase
coupled assay with NADH spectrophotometric monitoring at 340 nm)29
with tyrosine as substrate demonstrated that there was a total of 1200
units of lyase activity in the final preparation, which was stored at -80
°C in 1-mL aliquots.
Synthesis of Fluorotyrosine Analogues (2-10). For the standard
reaction, an aqueous solution (1 L) of fluorophenol (10 mM), pyruvic
acid (60 mM), pyridoxal-5′-phosphate (10 mg/L), ammonium acetate
(30 mM), â-mercaptoethanol (5 mM), and 30 units of tyrosine phenol-
lyase (TPL) was adjusted to pH 8 with dilute NH4OH and stored in
the dark at room temperature for 3-4 days.14 In the case of
tetrafluorophenol, 150 units of tyrosine phenol-layse was added, and
the reaction was allowed to proceed for 2-3 weeks at room temperature.
The mixture was then acidified with acetic acid (to achieve pH 3) and
filtered through a pad of Celite. The filtrate was extracted with 500
mL of ethyl acetate to remove unreacted fluorophenol. A prewashed
(6 N HCl, 6 N NaOH, water) cation exchange column (20 g of Dowex-
50W) was loaded with the reaction mixture, and the column was washed
with 5 column volumes of water followed by elution with 10% NH4OH
(aqueous). Fractions that gave a positive ninhydrin reaction were
combined and lyophilized to give a white solid, which was shown to
be pure fluorotyrosine analogue (0.5-2 g of product). Each analogue
was characterized using 1H NMR, 19F NMR, and HRMS with the data
shown below:
2-Fluorotyrosine (2). 1H NMR (D2O); δ 7.00 (brs, 1 H), 6.54 (brs,
2 H), 3.99 (m, 1 H), 3.90 (m, 1 H), 2.74 (m, 1 H). 19F NMR (D2O);
δ -116 (t, J ) 10 Hz, 1 F). HRMS calcd. for C9H10NO3F (MH)+,
200.0723; found, 200.0723.
3-Fluorotyrosine (3). 1H NMR (D2O); δ 7.09 (d, J ) 11.7 Hz, 1
H), 6.98 (m, 2 H), 3.94 (dd, J ) 8.0, 5.1 Hz, 1 H), 3.21 (dd, J ) 14,
5.1 Hz, 1 H), 3.05 (dd, J ) 14, 8.0 Hz, 1 H). 19F NMR (D2O); δ
-137 (m, 1 F). HRMS calcd. for C9H10NO3F (MH)+, 200.0723; found,
200.0721.
Synthesis of Fmoc-fluorotyrosine Analogues. General. The
fluorotyrosine analogue (1 mmol) was dissolved in 10% w/w aqueous
sodium carbonate (10 mL), and a solution of 9-fluorenylmethyl
N-succinimidyl carbonate (0.337 g, 2 mmol) in dioxane (5 mL) was
added dropwise while stirring the mixture at room temperature.
Subsequently, the reaction mixture was poured into water (50 mL) and
extracted with diethyl ether (2 × 100 mL). The aqueous phase was
acidified while being vigorously stirred with concentrated HCl to reach
pH 3, and then the aqueous phase was extracted with ethyl acetate
(100 mL). The organic phase was dried over anhydrous MgSO4, and
the solvent was removed in vacuo. The Fmoc derivative whose identity
was verified by TLC and NMR was used in peptide synthesis without
further purification.
Synthesis of Heptapeptides Containing Fluorotyrosine Analogues.
Heptapetides (EDNEXTA) with X ) tyrosine analogues 1-10 were
synthesized by automated Fmoc solid-phase peptide synthesis on a 0.1-
0.25 mmol scale using the unprotected Fmoc-fluorotyrosine analogues
generated above. The peptides were purified on a C-18 reverse phase
column using a linear gradient (acetonitrile/water containing 0.05% v/v
trifluoroacetic acid) and then were lyophilized to dryness. The
molecular weight of each heptapeptide was confirmed using electrospray
mass spectrometry, and purity (>95%) was determined by analytical
HPLC.
Profiles of pH versus Rate for Csk-Catalyzed Phosphorylation
of the Heptapeptide Family EDNEXTA (X ) 1, 6, 8-10). Kinase
assays were performed using the spectrophotometric coupled assay that
monitors ADP formation with minor modifications of previously
described procedures.24 Briefly, reactions contained 60 mM buffer,
15 mM MgCl2, 1 mM ATP, 190 µM NADH, 1 mM phosphoenolpyru-
vate, excess pyruvate kinase and lactate dehydrogenase, and pure
recombinant Csk. At least five different peptide substrate concentrations
in the range of 1-16 mM were used, and duplicate assays gave rate
2,6-Difluorotyrosine (4). 1H NMR (D2O); δ 6.22 (m, 2 H), 3.67
(m, 1 H), 3.01 (dd, J ) 14.8, 6.0 Hz, 1 H), 2.88 (dd, J ) 14.8, 8.0 Hz,
(29) Chen, H.; Gollnick, P.; Phillips, R. S. Eur. J. Biochem. 1995, 229,
540-549.

6858 J. Am. Chem. Soc., Vol. 120, No. 28, 1998
Kim and Cole
constants within 20%. Turnover of the limiting substrate was not
allowed to exceed 10%. Data were fit to the Michaelis-Menten
equation using a nonlinear curve fit, which in a number of cases only
allowed accurate kcat/Km values to be obtained because the Km values
were larger than 10 mM. Buffers used were Bis-Tris for pH 6.0-6.3,
Mops for pH 6.3-7.5, and Tris for pH 7.5-8.6. Although at pH 7.5,
an overlap point for Mops and Tris, the kcat/Km with Mops tended to
be higher (1.2-2-fold) than with Tris for the various peptide substrates;
normalization did not lead to a significant impact on the calculated
acidity constants. Enzyme activity was stable (<10% activity loss)
over all pH values used in this study for at least 5 min. Reactions
were initiated with Csk, and reaction rates were linear for at least 5
min at all pH values. Plots of log(kcat/Km)app vs pH for EDNEYTA
were fit to:
C-18 column using a linear gradient of acetonitrile-water containing
0.05% trifluoroacetic acid. Unphosphorylated peptides showed reten-
tion times of approximately 24 min and phosphorylated peptides
retention times of approximately 16 min. The molecular composition
of phosphopeptides was confirmed using electrospray mass spectrom-
etry.
Reverse Kinase Assays. The general procedure involved a direct
HPLC assay monitoring of the conversion of phosphopeptide to
dephosphopeptide. Using this approach, the lower limit of detection
of the conversion of peptide to dephosphorylated peptide was 0.5%
turnover. Standard reaction conditions were performed with 60 mM
Tris-HCl, pH 7.4, 12 mM MgCl2, 1 mM ADP, and 1 mM DTT at 30
°C in 40 µL. A 20-µL aliquot was diluted with 40 µL of water, and
the sample was injected onto an HPLC (C-18 column) with flow rate
1 mL/min using a linear gradient (acetonitrile-H2O with 0.05%
trifluoroacetic acid) as above with UV monitoring at 214 nm. Peak
areas of peptides were determined by integration using the Rainin
software package and referenced against standard amounts of pure
materials. The true reverse kinase activity was obtained by subtracting
(where necessary) the rate of phosphoryl transfer in the absence of
ADP from the total rate (in the presence of ADP). Subtraction proved
to be necessary for peptides with fluorotyrosine analogues 1, 2, 7, and
8, which had reproducible background rates of 40%, 70%, 60%, and
40% of the total rates, respectively. However, the overall slope in
Figure 6 (-0.33 ( 0.06) was not significantly affected by omission of
these correction factors (-0.27 ( 0.05) or by complete omission of
the points with high backgrounds (-0.38 ( 0.05). For all other
peptides, the background rates were <15% of the total rates. All rate
measurements were performed at least three times, and repeat measure-
ments gave rates within 20% of each other. Reactions were shown to
display linear activity for at least 60 min, and activity was linear with
respect to Csk enzyme concentration in the 1-10 µM range. Reaction
rates were linear with respect to phosphopeptide concentration range
(1-4 mM) for all phosphopeptides so that only kcat/Km results are
plotted. Initial conditions were used for all rate measurements with
less than 10% turnover of the limiting substrate.
log(kcat/Km)app ) log({kcat/Km}/[1 + H/K1])
where H is the proton concentration and K1 is the dissociation constant
for the ionizable group that is active when deprotonated. For
EDNEXTA where X ) fluorotyrosine analogue, the equation used was
log(kcat/Km)app ) log({kcat/Km}/[1 + H/K1 + K2/H])
where H is the proton concentration, K1 is the dissociation constant
for the ionizable group that is active when deprotonated, and K2 is the
dissociation constant for the ionizable group that is active when
protonated. Fits employed the nonlinear least squares approach in the
Kinetasyst II (Intellikinetics) software package, and dissociation
constants ( standard errors are shown in Figure 4. The calculated
pH-independent values of kcat/Km were associated with significant
standard errors (>50%) for peptides containing fluorotyrosine analogues
8-10. This error along with the decreased number and more limited
spread of data points prohibited an accurate determination of the ânuc
using these pH-independent kcat/Km values. In any event, the ânuc that
could be estimated using these data was near 0 as previously determined.
Preparation of O-Phosphofluorotyrosine Heptapeptides. The 10
different O-phosphofluorotyrosine peptides were prepared by large-
scale enzymatic phosphorylation of peptides EDNEXTA (X ) 1-10)
with Csk. The standard reaction conditions on a 1-mL scale included
the following: 60 mM Tris-HCl, pH 7.4; 3 mM ATP; 3 mM
phosphoenolpyruvate; 3 mM MnCl2; 1 mM peptide; 5 mM DTT; 10
µg PK-LDH (3:1 pyruvate kinase-lactate dehydrogenase, Boerhinger
Mannheim); and 1 µM Csk at 30 °C. For the peptide containing
tetrafluorotyrosine (10), the reaction was done using MOPS-Na buffer,
pH ) 6.6, with 3 µM Csk. Reactions were followed by analytical
reverse-phase HPLC and generally stopped after 48 h where 60-90%
conversion was observed. Approximately, 30 1-mL reactions were
performed in parallel for each peptide. Purification of phosphorylated
peptides (>95%) was accomplished using reverse-phase HPLC with a
Acknowledgment. Support from the Damon Runyon Scholar
Award Program, the NIH (CA 74305-01), the Irving Hansen
Foundation, and the Monique Weill-Caulier Trust is gratefully
acknowledged. We thank R. Phillips for the tyrosine phenol-
lyase overproducing strain and helpful advice. We thank A. S.
Mildvan for helpful suggestions and a critical evaluation of the
manuscript.
JA9808393