Chemical influences on the specificity of tyrosine phosphorylation.

Biological tyrosine phosphorylation has become an extensively studied reaction. Little, however, is known of the chemistry involved. The acetylation of the tyrosyl phenolic hydroxyl group by N-acetylimidazole was studied as a model acylation reaction over the pH range 7.5-9.5. The reactivities of tyrosine and 3-fluorotyrosine were compared. The ratio of reactivities, kappa F-Tyr/kappa Tyr, decreases with increasing pH. Extrapolation to the state in which equivalent concentrations of the two derivatives exist indicates that, consistent with Brønsted theory, tyrosine is 17 times more reactive than fluorotyrosine. No reactivity was observed with tetrafluorotyrosine, 3-nitrotyrosine, or 3,5-dinitrotyrosine. A peptide containing fluorotyrosine was synthesized and compared with the tyrosine-containing peptide as a substrate for the insulin receptor/tyrosine kinase. In both the presence and absence of insulin, the tyrosine peptide was phosphorylated with higher Vm and Km values than the fluorotyrosine peptide was. These results suggest that ionization of the tyrosyl hydroxyl group has an effect on both the chemical and enzymatic reactivities of the tyrosyl residue in acylation reactions. A model is suggested in which deprotonation of the acceptor occurs upon binding of the substrate to the kinase and implicates a role for the substrate site microenvironment in defining substrate specificity.

Biological tyrosine phosphorylation has become an extensively studied reaction. Little, however, is known of the chemistry involved.
The acetylation of the ty-rosy1 phenolic hydroxyl group by iV-acetylimidazole was studied as a model acylation reaction over the pH range 7.5-9.5.
The reactivities of tyrosine and 3-fluorotyrosine were compared. The ratio of reactivities, k m&Tyr, decreases with increasing pH. Extrapolation to the state in which equivalent concentrations of the two derivatives exist indicates that, consistent with Brensted theory, tyrosine is 17 times more reactive than fluorotyrosine.
A peptide containing fluorotyrosine was synthesized and compared with the tyrosine-containing peptide as a substrate for the insulin receptor/tyrosine kinase. In both the presence and absence of insulin, the tyrosine peptide was phosphorylated with higher V,,, and K, values than the fluorotyrosine peptide was. These results suggest that ionization of the tyrosyl hydroxyl group has an effect on both the chemical and enzymatic reactivities of the tyrosyl residue in acylation reactions. A model is suggested in which deprotonation of the acceptor occurs upon binding of the substrate to the kinase and implicates a role for the substrate site microenvironment in defining substrate specificity.
Tyrosine phosphorylation is an integral aspect of cellular growth and transformation (I-4). A major emphasis in the field involves characterization of the substrate specificity displayed by tyrosine kinases. Many protein kinases, including the CAMP-dependent kinase, recognize phosphorylation sites within specific primary sequences. Tyrosine kinases also Tyrosine kinases also seem to require a high order structure (11) near the phosphorylation site. A preliminary investigation indicates requirements for some additional structural features, possibly including a P-turn (11) near the phosphorylation site. Little, however, is known of the chemical features involved in the acylations of tyrosine residues. We have approached this problem by investigating the effect of substitution in the aryl ring of tyrosine on the reactivity of the tyrosyl hydroxyl group. Primarily fluorine derivatives were utilized because fluorine substitution enables a highly electronegative center to be introduced without steric factors. Specifically, we have examined the chemical and enzymatic modification of substituted tyrosine derivatives. Acetylation of the hydroxyl group of various ring-substituted tyrosines by N-acetylimidazole (12) was examined as a model acylation reaction. The results demonstrate a dependence on the pK, of the reacting phenolic group and are consistent with Brensted theory. These results have been used to interpret the phosphorylation of peptides containing tyrosine or fluorotyrosine by the insulin receptor/ kinase and provide insight into the mechanism of the tyrosine kinase reaction. Kirk's protocol was followed through the silica gel step. Following this, L-F-Tyr was isolated by preparative thin layer chromatography on fluorescent-backed silica plates. The developing system consisted of 1-butanol, acetic acid, water, and pyridine in a ratio of 15:3:12:10. The bands containing F-Tyr were excised, and the F-Tyr was extracted with ethyl ether. The extracted material was dried to an orange-brown viscous liquid, decolorized, and loaded onto Sephadex G-10. The material was eluted with water and the fractions identified by UV-visible spectrophotometry.
The purity of the F-Tyr material was evaluated by thin layer chromatography, high performance liquid chromatography, and amino acid an&y& employing DL-F-Tyr as a standard.
The compound was also analyzed bv 'H NMR on a Bruker WM-300 spectrometer. -_

AND DISCUSSION
The acetylation of the phenolic hydroxyl group by Nacetylimidazole is a convenient model reaction for biological phosphorylation.
Both reactions are nucleophilic attacks on an electron-rich site in an acyl functional group.
At pH values of 7.5,8.5, and 9.5 only tyrosine and 3-fluorotyrosine reacted with NAI.
The reaction followed second-order kinetics under the conditions utilized.
No reaction was observed with tetrafluorotyrosine, 3-nitrotyrosine, or 3,5-dinitrotyrosine. The ratios of rate constants evaluated for these reactions are collected in Table  I   The substrate concentration was varied from 0.5 K, to 5. 0 K,,,. where [AIF-~yrl[AITyr = 1.0 (both fully deprotonated), the ratio kT,,/kF.,,, becomes equal to 17. This reveals that, consistent with Brensted theory, tyrosine is 17-fold more reactive than fluorotyrosine under conditions at which the hydroxyl group of each compound is fully deprotonated.
The seemingly higher reactivity of F-Tyr at pH 7.5 and 8.5 is because of the higher concentrations of reactive (anionic) species at these pH values. Assuming a similar relationship between tyrosine and tetrafluorotyrosine (pK, 5.40), analysis suggests that tyrosine would be 83-fold more reactive than tetrafluorotyrosine at pH 7.5. This may explain the lack of reactivity of tetrafluorotyrosine with NAI at pH 7.5. The phosphorylation of tyrosyl-containing peptides by the insulin receptor/kinase is also modulated by chemical features of the acceptor substrate. Peptides containing either tyrosine or fluorotyrosine were used as substrates for the insulin receptor/tyrosine kinase. Both the tyrosyl-and fluorotyrosylcontaining peptides were phosphorylated in both the absence and presence of insulin; insulin stimulated peptide phosphorylation 3-4-fold. Replacement of the target residue by a derivative containing a hydroxyl group with a lower pK,, 9.21 uersus 10.07, dramatically affects the phosphorylation of the peptide. The fluorotyrosyl peptide was phosphorylated with an approximately 3-fold lower K,,, value, but the V, value for the reaction was decreased more than 8-fold in the presence of insulin. The kinetic parameters for both reactions are presented in Table II.
These results indicate that the ionization state of the hydroxyl group may be an important regulatory feature in both the binding of substrate and the phosphorylation reaction. The substrate with the lower pK, seems to bind better but is phosphorylated less well. As a first approximation, these results suggest that the enzyme has higher affinity for the anionic form of the substrate. By the same criterion, it seems unclear why, if the enzyme has higher affinity for the anionic species, the tyrosine peptide is phosphorylated with a higher V,,,. This result is consistent, however, with the results of the model acylation reaction provided the anionic species is formed upon binding to the enzyme. When bound to the kinase, the hydroxyl group of each peptide is deprotonated equally well. It is predicated that once deprotonated, tyrosine will be more reactive, as in the model reaction.
Formation of a deprotonated seryl residue has been postulated for the phosphorylation of Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) by the CAMP-dependent protein kinase (21, 22) via general base catalysis. The deprotonation is proposed (21) to occur simultaneously with binding of the substrate to the enzyme and to utilize aspartic acid at position 184 of the catalytic subunit in the deprotonation of the serine residue (22). Because this aspartic acid is conserved in all protein kinases (23), deprotonation of the acceptor residue may be a feature of catalysis common to all protein kinases, with the deprotonated species being the reactive nucleophile. Although tyrosine, with pK, = 10.07 (16) uersus 13.6 (24) for serine, should be more easily deprotonated than serine is, the alkoxide of serine should be a more potent nucleophile than the phenoxide of tyrosine is. The chemical difference of the substrates may contribute to the lower V, value seen for tyrosine kinases in comparison with serine kinases. Cyclic GMP-and AMP-dependent protein kinases, for example, have values from 1000 to 4000 nmol/min/mg (25,26), but the insulin receptor kinase has values from 6 to 72 nmol/min/mg (27)(28)(29). Generation of a reactive anionic form of the substrate on the enzyme surface is not unknown, and it has been demonstrated that a thiolate anion is generated at the active site of glutathione transferase (30). It was suggested that the enzymes's binding energy is used to position the thiol in such a way to induce a decrease in the pK, of the thiol. Binding of the fluorotyrosyl peptide may have a lower K,,, because the presence of a more easily deprotonated hydroxyl requires a lower expenditure of binding energy. Although sharing common features in catalysis, tyrosine and serine kinases do not have overlapping specificities. For the insulin receptor/kinase, serine and threonine substrate analogues serve as inhibitors of the tyrosine kinase activity (31). The Km values of the peptide substrates are approximately 2 mM; each of the inhibitory peptides has a K, of the same magnitude (2-4 mM). Because the rest of the peptide is identical, it is clear that the kinase must be able to distinguish the tyrosine from serine and threonine. Similar discrimination can be demonstrated with other kinases. Autophosphorylation of the tyrosine kinase, P130gag~fps, was completely blocked by mutation of tyrosine to serine or threonine (32). Enzymic functional groups other than the conserved aspartic acid residue must be involved to provide additional determinants of specificity.
This study suggests another aspect to the regulation of the substrate specificity of tyrosine kinases, particularly the insulin receptor/kinase.
Previous studies (5-10) have provided evidence that acidic residues on the N-terminal side of the modified tyrosine residue are important determinants of kinase specificity. The results of the present study demonstrate that the chemical environment about the phenolic hydroxyl group is an important feature in the specificity of tyrosine kinases. We propose that, in good substrates, the acceptor tyrosine residue is situated within an environment enabling efficient deprotonation of the tyrosine residue. The importance of the P-turn structure (11) may be related to the generation of the proper environment.