(Received for publication, August 21, 1995; and in revised form, October 10, 1995)
From the
We have addressed the question of whether protein kinase
substrate efficacy is a reliable barometer for successful inhibitor
design by assessing the dependence of k and k
/K
for eight
separate alcohol-bearing residues on solvent viscosity. We have found
that the K
for three structurally
distinct primary alcohol-containing peptides overestimates the affinity
that these species exhibit for the cAMP-dependent protein kinase. In
all three cases, the rate-determining step is product release, and
substrate binding is best described as rapid equilibrium. In contrast,
peptides containing the following phosphorylatable residues all provide K
values that are accurate assessments of
substrate affinity for the protein kinase: a secondary alcohol, a
simple phenol, and a primary alcohol with a relatively long side chain.
In the latter three instances, the rate-determining step is phosphoryl
transfer. Finally, two aromatic alcohol-containing residues that
possess lipophilic side chains exhibit Michaelis constants that
underestimate enzyme affinity. These results demonstrate that while it
may be tempting to employ structural elements from the most efficient
substrates (e.g. primary alcohols) for inhibitor design, less
effective substrates may serve as a more accurate assessment of
inhibitory success.
Protein kinases are often classified by their ability to
specifically phosphorylate tyrosine or serine/threonine residues in
intact proteins(1) . In general, protein kinases will utilize
aromatic or aliphatic alcohol-containing amino acids as substrates, but
not both (although the dual specificity kinases are
exceptions)(2) . However, we have found that this restriction
in substrate specificity vanishes in the context of non-amino acid
residues(3, 4, 5, 6, 7, 8, 9, 10, 11) .
For example, the cAMP-dependent protein kinase (PKA) ()catalyzes the phosphorylation of a wide assortment of
synthetic aliphatic and aromatic alcohols(3, 7) . In
addition, the ``tyrosine-specific'' protein kinases v-Abl and
c-Src exhibit an analogous broad active-site substrate
specificity(10, 11) . Interestingly, although the
active sites of these enzymes are unexpectedly tolerant, they are not
entirely indiscriminate. In the kinases examined to date, we have found
that each enzyme exhibits its own unique ability to recognize and
accommodate specific structural motifs associated with the
alcohol-bearing residue(8, 9, 11) .
Consequently, even such closely related enzymes as PKA and its
cGMP-dependent counterpart can be distinguished by their own unique
active-site substrate specificities(9) . Indeed, on the basis
of these results, we recently designed an affinity label that
specifically inactivates cGMP-dependent protein kinase, yet is
completely ineffective against PKA. (
)We have found
analogous discrepancies in the active-site substrate specificities of
v-Abl and c-Src as well(11) . In short, these unanticipated
differences in substrate specificity should significantly enhance the
ability to design agents targeted for specific members of the protein
kinase family. In addition, substrate specificity studies provide a
detailed assessment of the range of functionality that can be
accommodated within the active sites of protein kinases. This provides
a structural foundation upon which the design of inhibitors (e.g. transition state analogs, mechanism-based inhibitors, simple
ground state analogs, etc.) can be based.
PKA is the best understood
of all protein kinases, both in terms of structure and catalytic
behavior. This enzyme catalyzes the phosphorylation of a wide variety
of protein and peptide substrates, including the heptapeptide
Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide)(12) . The latter is an
extremely impressive substrate (K= 16 µM and V
=
20 µmol/min/mg). Although replacement of the serine moiety with a
nonphosphorylatable alanine provides a competitive inhibitor (i.e. Leu-Arg-Arg-Ala-Ala-Leu-Gly with respect to Kemptide), the
inhibitory efficacy of this species is decidedly unimpressive (K
= 320 µM) (13, 14, 15, 16) . The poor
inhibitory performance of the Ala-based peptide may be a consequence of
the missing hydroxyl moiety, which could play a critical role in
promoting peptide affinity. However, equilibrium dialysis measurements
of radiolabeled Kemptide (in the absence of ATP or in the presence of
AMP-PNP to preclude peptide phosphorylation) revealed a lackluster
affinity for PKA (K
= 210
µM)(13) . A recent series of experiments by Adams
and Taylor (17) has uncovered the explanation for the
discrepancy between the K
of Kemptide and
the K
and K
values obtained for it and analogous peptides, respectively.
These investigators employed viscosogenic agents to assess the
diffusion limits of the PKA-catalyzed phosphorylation of several
peptides. They established a minimum Kemptide K
for the PKA
ATP binary complex, a value that is
significantly larger than the corresponding K
. This difference between K
and K
values is due, in part, to an extremely rapid phosphoryl
transfer step. Unfortunately, this divergence in K
and K
is somewhat
disconcerting given the desire to relate the results of substrate
specificity studies to inhibitor design. What structural parameters
provide K
values for substrates that are
accurate indicators of enzyme affinity? We have addressed this question
by analyzing the effect of solvent viscosity on the rate of the
PKA-catalyzed phosphorylation of eight structurally distinct
alcohol-containing peptide substrates. We have found that with aromatic
and secondary alcohols, the K
is a
realistic indicator of PKA affinity. Indeed, in the case of two
aromatic substrates, the K
appears to be
an underestimate of how well the substrate binds to the enzyme active
site. In contrast, primary alcohol-containing substrates (with one
exception) provide K
values that
substantially overestimate PKA affinity.
We have recently begun to explore the active-site substrate specificity of protein kinases(3, 4, 5, 6, 7, 8, 9, 10, 11) . The results of these experiments provide a structural foundation upon which inhibitor design can be based. Namely, the range of functionality that can be readily accommodated within the active sites of these enzymes provides an assessment of what structural limitations, if any, exist with respect to inhibitor design. Unfortunately, substrate specificity studies do not necessarily provide a gauge of how specific structural motifs associated with the substrate influence affinity for the enzyme. We have now explored this issue with eight distinct structural archetypes, positioned on peptides at the site of phosphorylation.
There are a number of ways to examine the
relationship between the Michaelis constant of a particular substrate
and the enzyme affinity of that substrate. Obviously, the latter can be
directly assessed via such methods as equilibrium dialysis. However,
methods of this sort have the disadvantage that ATP cannot be employed
under the conditions of the experiment since the peptide substrate
would suffer phosphorylation. In the absence of ATP (or in the presence
of nonhydrolyzable ATP analogs), the K values
acquired may not provide an accurate estimate of the affinity of the
peptide for the protein kinase. Alternatively, nonphosphorylatable
substrate analogs can be examined with respect to their inhibitory
potency. However, these species lack the phosphoryl-accepting hydroxyl
moiety, a functional group that may very well play a key role in
augmenting the affinity of the substrate for the target enzyme.
Recently, Adams and Taylor (17) employed viscosogenic agents to
evaluate the individual microscopic rate constants associated with the
PKA-catalyzed phosphorylation of peptide substrates. Although this
method does not directly measure the K
for peptide
substrates, it does provide an assessment of the relationship between K
and enzyme affinity in the presence of
ATP.
The kinetic mechanism of PKA-catalyzed phosphorylation of
peptide substrates is (predominantly) an ordered sequential pattern,
with ATP binding first(17) . Product release is ordered as
well, with phosphopeptide discharged first, followed by ADP. The
kinetic pattern illustrated in Fig. S1describes the protein
kinase-catalyzed reaction under conditions of saturating ATP. The rate
of diffusion of peptide substrate into and out of the active site is
given by the microscopic rate constants k and k
, respectively. We have followed the
example of Adams and Taylor (17) by describing the rate of
product release with a single constant, namely k
.
Of the two products, the release of ADP is rate-determining. These
three rate constants are dependent upon solvent viscosity since this
parameter controls the rate at which agents enter or leave the enzyme
active site. In contrast, the rate constant (k
)
for the phosphoryl transfer step should be independent of solution
viscosity.
Figure S1: Scheme 1.
The slope ((k)
)
of the line generated from k
°/k
(
)as
a function of relative viscosity is given by .
If product release is rate-limiting (i.e. k
k
), then k
will
be strongly dependent upon solvent viscosity (i.e. (k
)
will be 1). In
contrast, if the phosphoryl transfer step is rate-limiting, then k
will be independent of solvent viscosity (i.e. (k
)
will be
zero). In an analogous fashion, the slope
((k
/K
)
) of
the line generated from (k
/K
)°/(k
/K
)
as a function of solvent viscosity is given by .
If the dissociation of the peptide substrate is slow compared
with phosphoryl transfer, then k/K
will be strongly
dependent upon relative viscosity. Under these circumstances, the
peptide substrate undergoes phosphorylation much faster than it
dissociates from the Michaelis complex. Substrates of this sort are
said to be catalytically committed or ``sticky''. In
contrast, if the phosphoryl transfer step is slow relative to substrate
dissociation, then k
/K
will
be independent of solvent viscosity. Table 1summarizes the
relative magnitude of the microscopic rate constants in terms of the
dependence of the steady-state kinetic parameters on solution
viscosity. Finally, the K
for the peptide
substrate is given by .
Consequently, the relative magnitudes of K and K
can be derived for the four extreme
conditions listed in Table 1. We note that a number of
assumptions (and controls) are required for a study of this nature.
These have been previously described and will not be explicitly dealt
with here(17) .
The eight peptide substrates employed in
this study are listed in Table 2Table 3Table 4. The
efficacy (k/K
) of these
compounds as substrates for PKA varies over nearly 4 orders of
magnitude (cf.3 and 6). Most of these species
are enzymologically as well as structurally distinct. Consequently,
they provide a relatively broad portrayal of the fashion by which
various substrates can interact with the active site of a protein
kinase.
The (k)
value of
0.99 ± 0.03 for the serine amide substrate 1 indicates
that the rate of release of ADP is slow relative to the phosphoryl
transfer step. Furthermore, the viscosity independence of k
/K
is evidence that the
phosphoryl transfer step is slow relative to the rate of peptide
substrate dissociation (k
). The latter is
characteristic of rapid equilibrium binding. In addition, these results
demonstrate that the K
for the serine amide
peptide 1 overestimates how well the substrate binds to PKA. The
fact that K
is not an accurate measure of enzyme
affinity for 1 is reminiscent of much of the earlier work with
Kemptide and related analogs(13, 14) . Indeed, Adams
and Taylor (17) extracted similar (k
)
and (k
/K
)
values for Leu-Arg-Arg-Ala-Ser-Leu-Gly in their viscosity studies.
The primary alcohol-containing peptides 2 and 3, like
their serine amide counterpart 1, exhibit a strong k dependence upon solvent viscosity. (
)Consequently, the release of ADP remains rate-limiting.
However, a somewhat different pattern has emerged for 2 and 3 with respect to the influence of viscosity on their k
/K
values. The leucinol
derivative 2 exhibits a modest dependence upon viscosity
((k
/K
)
= 0.23), whereas the corresponding cysteinol peptide 3 displays an even greater reliance upon this solution parameter
((k
/K
)
= 0.65). This represents a transition from a rapid equilibrium
(for 1) to a more catalytically committed status (for 2 and 3). In addition, it depicts a shift from a K
that is decidedly smaller than the K
to a situation in which the relationship between K
and K
becomes more
uncertain (i.e. as (k
/K
)
approaches unity; see Table 1). In the case of peptide 2, K
is given by
1.3(k
/k
)K
,
whereas for 3, the relationship is K
=
2.9(k
/k
)K
.
Since the slope of k
°/k
versus solvent viscosity is unity, it is clear that k
is significantly smaller than k
. Consequently, it is likely that for both
peptides 2 and 3, K
remains an
overestimate of substrate affinity for PKA.
In dramatic contrast to
the primary alcohol substrates in Table 2, peptides 4-6 (Table 3) all exhibit (k)
values at or near zero.
This is indicative of a phosphoryl transfer step that is slower than
product release. This, coupled with the fact that the slope of (k
/K
)°/(k
/K
) versus solvent viscosity is zero, indicates that the K
values obtained for substrates 4-6 are accurate estimates of the affinity that these species exhibit
for the protein kinase (Table 1). These substrates are a rather
diverse collection of functionality, containing secondary, primary, and
aromatic alcohols. However, they do share two nonstructural features,
namely, relatively high K
and relatively low k
values.
The k and k
/K
for substrates 4-6 are independent of solvent viscosity, an observation
that implies that the rate-determining step is phosphoryl transfer for
all three substrates. This is particularly noteworthy for the secondary
alcohol-containing substrate 4, which exhibits a structural
resemblance to threonine. Previously, we (19) as well as others (12, 20, 21) have shown that
threonine-containing peptides are notably poorer substrates than their
serine-containing counterparts. The K
values for
the former are significantly elevated relative to the latter. However,
it appears likely that these elevated K
values for
secondary alcohol-containing substrates are a more realistic assessment
of enzyme affinity than the Michaelis constants for primary alcohols
(such as those listed in Table 2). From the relationships between K
and K
provided in Table 1, this is due to a shift in the rate-determining step from
product release (primary alcohol substrates) to phosphoryl transfer
(secondary alcohol substrates).
Not all primary alcohols, however,
behave as those listed in Table 2. Phosphoryl transfer is
rate-limiting in the case of 5 (i.e. k is
nearly completely independent of solvent viscosity). This compound
contains a hydroxyl moiety three carbons removed from the peptide
backbone. In contrast, this distance is a mere two carbon atoms in
serine and related species (Table 2). Consequently, it is
possible that the alcohol functionality of 5 is inserted into
the active site in a fashion that is less than ideal for phosphoryl
transfer. Alternatively, the adjacent peptide bond, which likely plays
an important role in positioning the alcohol side chain in the active
site, may be improperly aligned. In either case, inappropriate
active-site/substrate alignment could result in a reduced rate of
phosphoryl transfer, thereby rendering this step rate-determining. In
either instance, both (k
/K
)
and (k
)
would be independent of
solvent viscosity.
The steady-state kinetic parameters of the
aromatic alcohol-containing peptide 6, like those associated
with 4 and 5, are viscosity-independent. In contrast, the
substrate efficacy of the corresponding aromatic alcohols 7 and 8 is clearly affected by the presence of viscosogenic agents.
The phosphoryl transfer step is fast relative to substrate
dissociation. In addition, the k for both
substrates is viscosity-independent, behavior emblematic of a
phosphoryl transfer step that is slow relative to product release.
These combined results indicate that the observed K
for 7 and 8 is an underestimate of the affinity of
these peptides for PKA (Table 1). As a consequence of the latter,
PKA affinity must be significantly larger for 7 and 8 than for 6. Finally, it is evident from Table 3and Table 4that peptides 7 and 8 are considerably more
efficient substrates than 6.
The relationship between the
microscopic rate constants k and k
for compounds 7 and 8 (k
k
) differs
from that of 6 (k
k
). There are two structural features that are
potentially responsible for this change in kinetic behavior. PKA
contains a lipophilic pocket adjacent to the enzyme active
site(22) . Compounds 7 and 8 possess a
hydrophobic tail positioned para to the phosphorylatable
alcohol, a structural feature missing in 6. In short, the
relatively slow substrate off-rate associated with the phosphorylation
of 7 and 8 may be due to enhanced binding augmented by
the attached hydrophobic substituents. Indeed, the S-benzylcysteinol derivative 3 also contains a
hydrophobic tail, and this species also exhibits a k
/K
that is strongly
viscosity-dependent, which likewise implies a slow substrate off-rate
compared with the rate of phosphoryl transfer. Furthermore, peptides 1, 4, and 5, which lack a hydrophobic substituent,
all exhibit (k
/K
)
values of zero. These results also imply that a hydrophobic residue at
the P + 1 position (or its equivalent) is important for promoting
the affinity of peptides for PKA. However, this change in the
relationship between the microscopic rate constants k
and k
can be
explained by a structural feature other than the lipophilic side chain.
Graves and co-workers (23) proposed several years ago that the
relatively low rate of turnover for tyrosine kinase-catalyzed
phosphorylations (versus those catalyzed by
serine/threonine-specific protein kinases) can be attributed to the low
nucleophilicity of the tyrosine hydroxyl compared with the aliphatic
alcohols of serine and threonine. In other words, the aromatic alkoxide
of 6 is inherently less reactive than the aliphatic alkoxides of 1-3. This could very well explain why the phosphoryl
transfer step is so slow for 6. In contrast, both 7 and 8 contain the electron-donating amide moiety, a structural
element that should enhance the nucleophilicity of the aromatic
alkoxide and thereby accelerate the rate of phosphoryl transfer.
Unfortunately, at this point, it is unclear whether the hydrophobic
side chains of 7 and 8 are responsible for a relatively
slow substrate off-rate or the electron-donating power of the amide
moiety should be held accountable for a comparatively rapid phosphoryl
transfer step. These two separate factors may very well be operating in
conjunction.
Others have previously shown that the Michaelis
constant and enzyme affinity are not necessarily identical for protein
kinase substrates. Adams and Taylor (17) have explicitly
demonstrated this with Kemptide. Walsh and co-workers (24, 25) have shown that the K for Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-Ile-amide is 4-fold
smaller than the K
for the corresponding
serine-containing substrate,
Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ser-Ile-amide. We have now shown
that the relationship between K
and enzyme
affinity is profoundly dependent upon the structural nature of the
alcohol-bearing residue. The primary alcohol-containing peptides 1-3 exhibit K
values that are smaller
than the estimated K
values. This observation may
be of some significance, particularly with respect to inhibitor design.
For example, an inhibitor that is structurally related to peptides 1-3 may very well exhibit a K
value
that is disappointingly higher than the observed Michaelis constants
for these substrates. In contrast, the secondary alcohol 4, the
primary alcohol 5, and the aromatic alcohol 6 furnish K
values that are accurate estimates of the
affinity that these peptides exhibit for PKA. Finally, the Michaelis
constants associated with the aromatic alcohols 7 and 8 appear to underestimate how well these substrates bind to the
enzyme active site. In short, although it is tempting to utilize the
most efficient substrates as the structural basis for inhibitor design,
less effective substrates may act as more accurate barometers of
inhibitory success.