©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Is Protein Kinase Substrate Efficacy a Reliable Barometer for Successful Inhibitor Design? (*)

(Received for publication, August 21, 1995; and in revised form, October 10, 1995)

Douglas S. Werner Tae Ryong Lee David S. Lawrence (§)

From the Departments of Chemistry and Medicinal Chemistry, Natural Sciences and Mathematics Complex, State University of New York, Buffalo, New York 14260

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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) (^1)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. (^2)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(max) = 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 PKAbulletATP binary complex, a value that is significantly larger than the corresponding K. This difference between Kand 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.


EXPERIMENTAL PROCEDURES

Materials

All chemicals were obtained from Aldrich, except for [-P]ATP (DuPont NEN), cAMP (Sigma), bovine serum albumin (Sigma), protected amino acid derivatives and Rink resin (Advanced ChemTech), and Liquiscint (National Diagnostics, Inc.). Dialysis tubing was purchased from Spectrum, and CM-Sephadex C-25 and Sephadex GS-100 (superfine) were obtained from Pharmacia Biotech Inc. SDS-polyacrylamide and Affi-Gel blue were purchased from Bio-Rad. Phosphocellulose P-81 filter papers were purchased from Whatman.

cAMP-dependent Protein Kinase Preparation

PKA was isolated and purified as described previously(18) .

Viscosity Buffer Preparation

Buffers with varying viscosities were prepared from glycerol (6, 15, 25, and 36 (w/w)) and sucrose (30% (w/w)) in 100 mM MOPS at pH 7.1. Relative viscosities were determined in triplicate with 3-ml samples using an Ostwald viscometer. The solutions were first equilibrated in a constant water bath at 30.0 °C for 5 min. The viscosities varied from 1.00 to 2.46 with glycerol to 2.64 with sucrose.

Peptide Synthesis and Purification

t-Butoxycarbonyl-Gly-Arg-Thr-Gly-Arg-Arg-Asn-(dimethoxydiphenylmethyl) was prepared on Kaiser's oxime resin with t-butoxycarbonyl-amino acids and subsequently displaced from the resin with a series of amino alcohols using previously described protocols(3) . The peptides were then deprotected with 90% trifluoroacetic acid, 10% anisole. The solvent was removed under reduced pressure, and the peptidic residue was dissolved in methanol and precipitated via addition of ethyl ether. The crude peptides were dissolved in a minimum of doubly distilled water and ion-exchanged on SP-Sephadex C-25 (0.4-1.2 M KCl gradient in 50 mM sodium acetate, pH 3.5; total volume of 400 ml). The partially purified peptides were subsequently subjected to preparative HPLC using three Waters radial compression modules (25 times 10 cm) connected in series: a gradient from 0 to 3 min (100% A), a linear gradient from 3 min (100% A) to 35 min (65% A and 35% B), and a linear gradient from 35 min (65% A to 35% B) to 40 min (30% A and 70% B). All peptides were subsequently reanalyzed by analytical HPLC using a Waters µBondapak C(18) column (3.9 times 300 mm). Those peptides that contained extraneous peaks were repurified on the analytical column. All of the purified peptides gave satisfactory fast atom bombardment mass spectral analysis.

Protein Kinase Assays

All assays were performed in triplicate at pH 7.1 and thermostated in a water bath maintained at 30 °C. Final assay volume totaled 50 µl and contained 100 mM MOPS, 150 mM KCl, 12.5 mM MgCl(2), 0.125 mg/ml bovine serum albumin, and between 0.35 and 175 nM catalytic subunit, depending on the efficiency of the substrate. For the determination of the kinetic constants, the following concentrations were employed: 100 µM [-P]ATP (100-500 cpm/pmol) and a substrate concentration over a 10-fold range around the apparent K(m). The phosphorylation reactions were initiated by addition of 10 µl of catalytic subunit diluted from a concentrated stock solution (1.5 mg/ml in 100 mM MOPS, 150 mM KCl, 1 mM dithiothreitol, and 0.125 mg/ml bovine serum albumin at pH 7.1). Reactions were terminated after 5.0 min by spotting 25-µl aliquots onto 2.1-cm diameter phosphocelluose paper discs. After 10 s, the discs were immersed in 10% glacial acetic acid and allowed to soak with occasional stirring for at least 1 h. The acetic acid was decanted, and the discs were washed with 4 volumes of 0.5% H(3)PO(4) and 1 volume of distilled water. A final acetone rinse was employed, and the discs were air-dried and placed in plastic scintillation vials containing 6 ml of Liquiscint prior to scintillation counting for radioactivity.

Determination of Kinetic Constants

The apparent K(m) (±S.D.) and V(max) (±S.D.) values were determined from initial rate experiments. The data from these experiments were plotted using the Lineweaver-Burk procedure, and the corresponding plots proved to be linear.


RESULTS AND DISCUSSION

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(d) 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(d) for peptide substrates, it does provide an assessment of the relationship between K(m) 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(2) 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(4). 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(3)) 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(^3)as a function of relative viscosity is given by .

If product release is rate-limiting (i.e. k(4) k(3)), 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(m))) of the line generated from (k/K(m))°/(k/K(m)) 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(m) 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(m) 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(m) for the peptide substrate is given by .



Consequently, the relative magnitudes of K(m) and K(d) 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(m)) 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(m) 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(m) for the serine amide peptide 1 overestimates how well the substrate binds to PKA. The fact that K(m) 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(m)) 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. (^4)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(m) values. The leucinol derivative 2 exhibits a modest dependence upon viscosity ((k/K(m)) = 0.23), whereas the corresponding cysteinol peptide 3 displays an even greater reliance upon this solution parameter ((k/K(m)) = 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(m) that is decidedly smaller than the K(d) to a situation in which the relationship between K(m) and K(d) becomes more uncertain (i.e. as (k/K(m)) approaches unity; see Table 1). In the case of peptide 2, K(m) is given by 1.3(k(4)/k(3))K(d), whereas for 3, the relationship is K(m) = 2.9(k(4)/k(3))K(d). Since the slope of k°/kversus solvent viscosity is unity, it is clear that k(4) is significantly smaller than k(3). Consequently, it is likely that for both peptides 2 and 3, K(m) 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(m))°/(k/K(m)) versus solvent viscosity is zero, indicates that the K(m) 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(m) and relatively low k values.

The k and k/K(m) 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(m) values for the former are significantly elevated relative to the latter. However, it appears likely that these elevated K(m) 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(m) and K(d) 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(m)) 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(m) 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(3) for compounds 7 and 8 (k k(3)) differs from that of 6 (k k(3)). 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(m) 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(m)) 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(3) 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(i) for Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-Ile-amide is 4-fold smaller than the K(m) 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(m) and enzyme affinity is profoundly dependent upon the structural nature of the alcohol-bearing residue. The primary alcohol-containing peptides 1-3 exhibit K(m) values that are smaller than the estimated K(d) 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(i) 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(m) 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.


FOOTNOTES

*
This work was supported by Research Grant GM45989 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 716-645-6800 (ext. 2170); Fax: 716-645-6963.

(^1)
The abbreviations used are: PKA, cAMP-dependent protein; AMP-PNP, 5`-adenylyl-beta,-imidodiphosphate; MOPS, 4-morpholinepropanesulfonic acid; HPLC, high pressure liquid chromatography.

(^2)
X. Yan, J. D. Corbin, S. H. Francis, and D. S. Lawrence, submitted for publication.

(^3)
The k° and (k/K)° terms are fixed, for each substrate, at 6% glycerol. The k and k/K terms vary as a function of solvent viscosity.

(^4)
We note that a few of the values for (k) and (k/K) are slightly greater than the theoretical maximum (1.0) or less than the theoretical minimum (0.0). The behavior is most likely a consequence of relatively subtle iterative errors generated during the experimental protocol.


ACKNOWLEDGEMENTS

We thank Professor Joseph Adams (Department of Chemistry, San Diego State University) for helpful advice.


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