Phosphorylation of Serine Induces Lysine pKa Increases in Histone N

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Phosphorylation of Serine Induces Lysine pKa Increases in Histone N-terminals and Signaling For Acetylation. Transcription Implications Lois Manning, and James M. Manning Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b01040 • Publication Date (Web): 15 Nov 2018 Downloaded from http://pubs.acs.org on November 16, 2018

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Biochemistry

Phosphorylation of Serine Induces Lysine pKa Increases in Histone N-Terminals and Signaling for Acetylation. Transcription Implications

Lois R. Manning and James M. Manning* Department of Biology Northeastern University Boston, MA 02115

*Corresponding Author: J.M. Manning Department of Biology Mugar Life Sciences Building Room 134 Northeastern University 360 Huntington Ave. Boston, MA 02115 Phone: 617-373-5267 Email: [email protected]

Supported in parts by NIH grants HL-15157, HL-18819 and HL-58512

Keywords: Histone, Acetylation, Phosphorylation, Nucleosome

Abstract

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The mild acetylating agent, methyl acetyl phosphate, is used to estimate the pKa values of some of the amine groups in peptides with sequences corresponding to a segment of the N-terminal tail of histone H4. When Ser-1 is not phosphorylated, the Lys epsilon amines have pKa values in the range of 7.8-8.3, which are much lower than currently assumed. When Ser-1 is phosphorylated, the pKa values of these Lys amines are elevated to the range of 8.8-10.3 thus providing the rationale for reports that they are then better substrates for acetyltransferases. Thus, reversal of suppressed pKa values of Lys epsilon amines by Ser phosphorylation represents the basis for signaling in histone Nterminal tails to promote hyperacetylation, which is a hallmark of transcriptionally active euchromatin. In contrast, a state of hypoacetylation is present in the absence of phosphorylation as in transcriptionally inactive heterochromatin. A novel approach for estimating pKa values based on a linkage between the Henderson-Hasselbalch and the Michaelis-Menten equations indicates that the pKa values of the Lys epsilon amines in H3 and H4 N-terminal tails have a highly variable charge gradient dependent on location and proximity to the phosphorylation site.

Introduction Acetylation of specific lysine sites in the amino terminal tails of histones has long been known to play a role in gene expression (1) but its mechanism has


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Biochemistry

been difficult to elucidate because of the number of such sites in each of the four core histones. Phosphorylation of other sites, particular serine, is known to be associated with this acetylation (2,3) but the details of this interrelationship are unclear. We have addressed these issues by studying the rates of chemical acetylation of synthetic peptides with only a single lysine available for acetylation and containing serine either free or phosphorylated. The N-terminal tail of histone H4 is relatively short-- consisting of 23 residues, contains the major Lys acetylation sites, is unstructured compared to the remainder of the highly structured histone H4 in the nucleosome core, and can be removed without major compromise to the histone core structure ( 4,5 ). Hence, it seems reasonable to assume that peptides containing tail sequences such as that used in this study would be a valid system to study acetylation details. This rationale has also been adopted by other investigators ( 6,7 ) who employed peptides corresponding to segments of the N-terminal tails of histone H3 to demonstrate that Ser-10 phosphorylation increases the efficiency ( decreased Km) of enzymic acetylation of nearby Lys-14 amine side chain; no mechanism was provided in those reports but, based on our studies here, we suggest one. In an earlier report (8), histone H3 peptides were studied and here we extend those results to histone H4 peptides; we demonstrate that both sets of results can be interpreted through a linkage between the Henderson-Hasselbalch and the Michaelis-Menten equations. We also present a method based on this relationship whereby pKa values can be estimated and we use it to demonstrate that the lysine epsilon amines in the amino-terminal tails of histones H3 and H4 have pKa values that are anomalously low when serine is unphosphorylated but are increased significantly when serine is phosphorylated, an example of signaling arising from a post-translational modification.

Materials and Methods Peptides- Peptides were synthesized at the Rockefeller University Protein/DNA Technology Center. Each peptide had the same 9-amino acid sequence ( SGRGKGGKG) corresponding to a segment of the N-terminal tail of histone H4 with different acetylation/phosphorylation patterns as shown in Table 1. When the free amino group at either Ser-1, Lys-5, or Lys-8 was studied, the other amines were blocked by complete acetylation during synthesis; Ser-1


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hydroxyl was either phosphorylated or remained free. The C-terminal carboxyl groups were amidated to block charge. After synthesis and purification, each peptide had the expected amino acid analysis after complete acid hydrolysis (Table 1). Mass spectrometry analyses were within 2.4 mass units ( 0.3%) of theoretical (887.5) for the unacetylated peptides and within 1.2 mass units ( 0.1%) of theoretical ( 966.4) for the acetylated peptides (Table 1). Chemical Acetylation- Methyl acetyl phosphate ( MAP ) was introduced by Kluger and colleagues ( 9, 10 ) as a very useful site-specific agent which, when judiciously employed at relatively low concentrations in the presence or absence of substrates or effectors that bind to the active site, provides unequivocal evidence for important protein amines. We prepared MAP as described by Kluger and colleagues ( 9). It is very stable over the course of the kinetic studies as judged by the linearity of the first-order profiles. Unlike potent acetylating agents such as acetic anhydride, MAP is a very mild reagent so that it is feasible to perform accurate kinetic studies (8). In the studies described below, we used it at concentrations of 10, 25, 50, 75, and 100 mM and followed the course of its reaction with a given peptide ( 0.2-0.3 mM as determined by amino acid analysis) for 10-15 minutes in Hepes buffer at pH 7.5 and 25 degrees; disappearance of the amine was followed with the Fluram reagent ( 8 ). Under such conditions, reaction progress with respect to formation of acetylated peptide was slow and the kinetic profiles were linear as semi-log plots of per cent peptide amine remaining with time so that accurate kinetic constants could be calculated. Denu and colleagues ( 11 ) have also studied non-enzymic lysine acetylation using acetyl-phosphate or acetyl-CoA. We have previously used MAP at relatively low concentrations to acetylate the major amino groups in the DPG binding pocket of hemoglobin (12-14). This was established by peptide mapping of tryptic digests followed by amino acid analysis and also by competition experiments with DPG and MAP; minor acetylation sites were also noted. Others have also used MAP in the presence or absence of substrates of a dehydrogenase to successfully label its active site ( 15). A claim that the selectivity of MAP was very broad employed very high MAP concentrations ( 16 ) at which preferential acetylation was likely obscured. Furthermore, acetylated sites in that study were not unambiguously assigned. Kozarich and colleagues ( 17, 18) have demonstrated the wide applicability of acyl phosphate monoesters linked to ATP to selectively label Lys side chains at the active sites of protein kinase families, and the ATP-dependent molecular chaperone heat shock protein 90.

Results and Discussion


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Biochemistry

Kinetic Profiles- The relationship between varying pH and reaction rates to determine pKa values for specific groups on proteins is well established. Hence, Westheimer and colleagues ( 19-21 ) reported an anonymously low pKa of 5.9 for the active site Lys of acetoacetate decarboxylase; this result was confirmed by site-directed mutagenesis and shown to be due to an adjacent Lys residue ( 22). Usually, in pH vs activity studies, buffers of varying pH are used to alter the ratio of protonated to unprotonated an active site group while its intrinsic pKa remains constant. Our studies described here and in a previous communication (8) focus on the effect of a change in ionization state of the substrate for acetylation whose pKa has changed as a result of serine phosphorylation at constant pH, i.e. in the former example, pH is variable and pKa is constant whereas in our studies, pH is constant and the pKa changes; in both cases the ratio of protonated to unprotonated side chain is affected.

The kinetic profiles of the chemical acetylation of peptides containing free amine groups at Ser-1, Lys-5, or Lys-8 using varying MAP concentrations are shown in Fig. 1 in their unphosphorylated and their phosphorylated forms (top panels A,C,E). The profiles indicate that for each peptide, the phosphorylated peptide reacts significantly slower with MAP than its unphosphorylated counterpart. The hyperbolic nature of these three plots resemble those of an enzyme-catalyzed reaction indicative of saturation kinetics. When the same data are plotted as double reciprocals ( lower panels B,D,F), the points fall on straight lines that resemble Lineweaver-Burk plots. It is obvious from both types of plots that the lines converge the greater the distance of the amine from the phosphate group. The data show that the rates decrease for the unphosphorylated peptides and increase for the phosphorylated peptides likely due to variations in the degree of charge on the amine. These results are consistent with a mechanism in which the peptide and MAP initially combine rapidly and reversibly to form a complex followed by slow irreversible acetylation (Fig. 2, Michaelis-Menten segment).


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Similar to Lineweaver-Burk plots for enzyme-catalyzed reactions, we calculated Vmax and Kb ( analogous to Km) for the chemical acetylation ( Fig.1, lower 3 panels, B,D,F). Vmax is the acetylation rate at infinite MAP concentration ( y-axis intercept value when 1/MAP = 0). Vmax values were similar for all


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Biochemistry

peptides ( Table 1 ), as is clear from a visual inspection of the Lineweaver-Burk plots which show that the lines cross the y-axis at similar points for all peptides ( red horizontal arrows). Vmax and Kb are related by: Kb + (MAP) = (Vmax) (MAP) / v, which is equivalent to the Michaelis-Menten equation for an enzyme-catalyzed reaction. When 1 / v = 0, where the blue lines intercept the negative x-axis, the right term cancels and the equation can be rearranged to Kb = - MAP, ( two red vertical arrows in each lower panel B,D,F); these represent Kb values in terms of MAP concentrations which are highly variable for each peptide ( Table 1 ). The red arrow pairs merge with one another as the distance from the phosphorylation site increases, which is an indication of the decreasing strength of the phosphorylation inductive effect.

Mechanism of Phosphorylation Effect on Acetylation- The chemical acetylation of the histone peptide amines by MAP involves the unprotonated form of the amine as shown by Kluger and colleagues ( 8 ). Both protonated and unprotonated forms are present although their ratio is a function of many factors. The data in Figure 1 indicate that there is less of this unprotonated form present when the peptide is phosphorylated, i.e. the -NH2/NH3+ ratio in the HendersonHasselbalch segment of Fig. 2 decreases relative to the ratio for the unphosphorylated peptide. We suggest that this decreased ratio arises from an increased pKa of the amine upon phosphorylation analogous to the elevation of

-

the amine pKa by the carboxyl group in Glycine, +H3N-CH2-COO, compared to Glycinamide, +H3N-CH2-CONH2, 9,7 vs 7.9, respectively ( 23, 24 ). This inductive effect by phosphate persists for a distance of 8-9 amino acid residues and decreases as the distance from the phosphate increases ( Fig. 1). This would be expected to lead to higher Kb values since more MAP would be needed to counteract the increased amount of protonated amine thus affecting MichaelisMenten kinetics and Kb values. Since the reaction rates shown in the upper panels of Fig. 1 show hyperbolic profiles indicating that lower concentrations of MAP lead to proportionally faster rates than higher concentrations do, repulsion of MAP by the phosphorylated peptide as a reason for higher Kb values appears unlikely. Ionization Effects-The direct effect of pH on Km values has been known for many years ( 24, 25); it can arise from several sources, such as the ionization state of the substrate, of the enzyme, or of the enzyme-substrate complex. The serine-O-phosphate induced elevation of the pKa of nearby amine groups focuses on another aspect to this relationship since it instantly changes the existing ratio of the unprotonated / protonated states of the amine group due to the Henderson-Hasselbalch equation, which then affects the degree of charge on the substrate for acetylation in the Michaelis-Menten equation (Fig. 2). Furthermore, since the -NH2/-NH3+ ratio is a logarithmic function, it represents a


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signal amplification factor perhaps contributing to the distance of the effect as far as 8-9 amino acids. The alkaline Bohr Effect in hemoglobin is another example of an induced pKa difference (His-146-beta) between the deoxy- and oxy states derived from their protein conformational switch contributing to either binding or release of protons and oxygen (27). In the present study, the induced pKa change arises from a post-translational modification. Enzymic vs Chemical Acetylation- Whereas chemical acetylation with MAP requires an unprotonated amine ( 8 ), a protonated amine is necessary in enzymic acetylation in order for the substrate to bind effectively to an acetyltransferase ( 28 ). Hence, the effect of phosphorylation on acetylation resulting in an increase in Kb for chemical acetylation would be expected to lead to a decreased Km, i.e. more efficient binding to the enzyme and hence increased acetylation. Indeed, this has been observed with the GCN5 histone acetyltransferase (6, 7) where the increased binding of the peptide phosphorylated at Ser-10 extends a considerable distance to Lys-14. These results are consistent with our earlier reports on chemical acetylation of H3 peptides where Ser-10 phosphorylation led to propagation of lysine basicities up to 9-10 residues on either side of the Ser-10 phosphorylation site (8).


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Biochemistry

pKa Estimates from Kb Values- The linkage between the MichaelisMenten and the Henderson-Hasselbalch equations shown in Fig. 2 suggests that the experimentally determined Kb values can be used to obtain estimates of a pKa when the third variable, pH, is known. We used the experimentally determined Kb values of glycinamide (Glyamide), +H3N-CH2-CONH2, and of glycine (Gly) +H3N-CH2-COO as standards to construct the x-axis of the line shown in Fig.3. Since the pKa values of Glyamide and Gly are known ( 7.9 and 9.7, respectively ( 23, 24 )), the ratio of -NH2 / NH3+ of each one at pH 7.5, which is the pH of the acetylation reaction, was calculated and used for the left yaxis; a straight line between them was drawn. These two pKa values are sufficiently different so that a relatively large span of unknown pKa values is covered. The Kb values of the histone peptides found in Figure 1( panels B,D,F ) and Table 1 were placed on the line as the x-axis values and the corresponding left y-axis values of NH2 / -NH3+ were read from the graph. From this ratio, the pKa for each peptide was calculated using the Henderson-Hasselbalch equation ( Table 1 and right Fig. 3 y-axis). We emphasize that these values are only relative estimates although significant differences between them are readily discernable. Such a graph is similar to that used for SDS-PAGE gels to determine an unknown subunit molecular weight by its migration distance compared to the migrations of standards with known molecular weights. The pKa values for all amines tested in the unphosphorylated N-terminal tails of histones H3 and H4 in this and an earlier report ( 8 ) are clustered in the


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range of 7.8-8.3 (Fig.3), which is significantly below the values of 10-11 usually assumed for the epsilon amines of Lys. However, we could not find published measurements to document such high values. It is well known that baseweakening groups such as Arg and Lys lowers the pKa values of adjacent bases such as the Lys epsilon amino group ( 29 ); the intact N-terminal tails of histones H3 and H4 contain 10 and 9 Arg + Lys residues, respectively, and no acidic side chains thus supporting the conclusion above that Lys pKa values are suppressed in unphosphorylated histones. Furthermore, simple diamines are known to have a lowered pKa value in the range shown here ( 30 ). In addition, Lys pKa values in proteins can vary widely ( pH< 6 to pH >11) due to local environment effects (31-33 ). The postulate of Westheimer and colleagues, referred to above, that the very low pKa of the active site Lys of acetoacetate decarboxylase was due to a second adjacent positively charged Lys ( 19-21 ) was confirmed and extended by Highbarger et al. using site-directed mutagenesis ( 22 ). Biological Implications of Variable pKa Values- The results in Figure 3 (right y-axis) for the pKa values of the lysine side chains of histone H4 peptides described here together with earlier results on lysines of histone H3 peptides ( 8 ) show that their amine groups have highly variable pKa values. A physiological consequence might be that with such low pKa values in the absence of phosphorylation, such lysines may not be fully protonated at physiological pH and would likely bind to aceyltransferases with decreased efficiency resulting in low extents of acetylation, i.e. hypoacetylated as in transcriptionally inactive heterochromatin. In contrast, the phosphorylated peptides in Fig. 3 and Table 1 have pKa values that are much higher and more widely distributed (pH 8.8-10.3) than their unphosphorylated counterparts. Such peptides may bind much tighter to acetyltransferases ( 6, 7 ) and thus would be more extensively acetylated as in hyperacetylated transcriptionally active euchromatin (34, 35 ). This model establishes a relationship between acetylation and phosphorylation in histones and suggest how biologically relevant hyperacetylation is accomplished.

References 1) Allfrey, V.G., Faulkner, R., and Mirsky, A.E. (1964) Acetylation and methylation of histones and their possible role in the regulation of RNA synthesis, Proc.Natl. Acad. Sci. USA, 51, 786-794. 2) van Holde, K.E. (1989) Chromatin, Springer-Verlag, New York. 3) Wolffe, A. (1998) Chromatin: structure and function, Academic Press, San Diego.


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Biochemistry

4) Ausio, J., Dong, F., and van Holde, K.E. (1989) Use of selectively trypsinized nucleosome core particles to analyze the role of histone "tails" in the stabilization of the nucleosome, J. Mol. Biol. 206, 451-463. 5) Widlund, H.R., Vitolo, J.M., Thiriet, C., and Hayes, J.J. (2000) DNA sequence dependent contributions of core histone tails to nucleosome stability: Differential effects of acetylation and proteolytic tail removal, Biochemistry 39, 3835-3841. 6) Lo, W.S., Trievel, R.C., Rojas, J.R., Duggan, R., Hsu, J.Y., Allis, C.D., Mamorstein, R. and Berger, S.L. (2000) Phosphorylation of Ser-10 in histone H3 is functionally linked in vitro and in vivo to acetylation at Lys-14, Mol Cell 5, 917926. 7) Clements, A., Poux, A.N., Pillus, L., Berger, S.L. and Marmorstein R. (2003) Structural basis for histone and phosphohistone binding by the GCN5 histone acetyltransferase, Mol.Cell, 12, 461-473. 8) Manning, L.R., and Manning, J.M. (2018) Contributions to nucleosome dynamics in chromatin from interactive propagation of phosphorylation /acetylation and inducible histone lysine basicities, Protein Science, 27, 662-671. 9) Kluger, R. and Tsui, W-C ( 1980) Methyl acetyl phosphate. A small anionic acetylation agent. J.Org.Chem. 45, 2723. 10) Wodzinska, J. and Kluger, R. (2008) pKa-Dependent formation of amides in water from an acyl phosphate monoester and amines, J.Org. Chem. 73, 47534754. 11) Baeza, J., Smallegan, M.J. and Denu, J. ( 2015) Site-specific reactivity of non-enzymic lysine acetylation, ACS Chem. Biol. 10, 122-128. 12) Ueno, H., Pospischil, M., Manning, J.M., and Kluger, R. (1986) Site-specific modification of hemoglobin by methyl acetyl phosphate, Arch. Biochem. Biophys. 244, 795-800. 13) Ueno, H., Pospischil, M.A., Kluger, R., and Manning, J.M. (1986) Methyl acetyl phosphate: a novel acetylating agent. its site-specific modification of hemoglobin A, J. Chromatog. 359, 193-201. 14) Ueno, H., Pospischil, M.A., and Manning, J.M. ( 1989) Methyl acetyl phosphate as a covalent probe for anion binding sites on human and bovine hemoglobins, J. Biol. Chem. 264, 12344-12351. 15) Kataoka, K., Tanizawa, K., Fukui, T., Ueno, H., Yoshimura, H., Esaki, N., and Soda, K. (1994), Identification of active site Lysine residues of phenylalanine dehydrogenase by chemical modification with methyl acetyl phosphate combined with site-directed mutagenesis, J. Biochem. 116, 1370-1376.


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16) Xu, A.S.L., Labotka, R.J. and London, R.E. (1994) Acetylation of human hemoglobin by methyl acetylphosphate, J.Biol. Chem. 274, 26629-26632. 17) Patricelli, M.P., Szardenings, A.K., Liyange, M., Nomanbhoy, T.K., Weissig, H., Wu, M., Aban, A., Chun, D., Tanner, S., and Kozarich, J.W. ( 2007) Functional interogation of the kinome using nucleotide acyl phosphates, Biochemistry 46, 350-358. 18) Nordin, B.E., Liu, Y., Aban, A., Brown, H.E., Wu, J., Hainley, A.K., Rosenblum, J.S., Nomanbhoy, T.K., and Kozarich, J.W. ( 2015) ATP acyl phosphate reactivity reveals native conformations of Hsp 90 paralogs and inhibitor target engagement, Biochemistry 54, 3024-3036. 19) Schmidt, D.E., and Westheimer, F.H. ( 1971) pK of the Lysine amino group at the active site of acetoacetate decarboxylase, Biochemistry, 10, 1249-1253. 20) Frey, P., Kokesh, F.C., and Westheimer, F.H. ( 1971), A reporter group at the active site of acetoacatate decarboxylase. I. Ionization constant of the nitrophenol, J. Amer. Chem. Soc., 93, 7266-7269. 21) Kokesh, F.C. and Westheimer, F.H. ( 1971), A reporter group at the active site of acetoacetate decarboxylase. II. Ionization of the amino group, J.Amer. Chem. Soc. 93, 7270- 7274.

22) Highbarger, L.A., Gerlt, J.A., and Kenyon, G.L. ( 1996) Mechanism of the reaction catalyzed by acetoacetate decarboxylase. Importance of Lysine 116 in determining the pKa of active-site Lysine 115, Biochemistry, 35, 41-46. 23) Cohn, E.J. and Edsall, J.T. (1943) Proteins, amino acids, and peptides as dipolar ions. Reinhold Publishing, New York, pg. 99. 24) Edsall, J.T. and Wyman, J. (1958) Biophysical Chemistry, Vol. 1, Academic Press, New York, pg. 208. 25) Dixon, M. (1953) The effect of pH on the affinities of enzymes for substrates and inhibitors. Biochem.J. 55, 161-170. 26) Cornish-Bowden, A. (1979), Fundamentals of enzyme kinetics, Butterworth. 27) Perutz,M., Gronenborn, A.M., Clore, G.M., Fogg, J.H., and Shih, D.T.B. (1985) The pKa values of two histidines in human hemoglobin, the Bohr Effect, and dipole moments of alpha helices, J.Mol.Biol. 183, 491-498.


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28) Dutnall, R.N., Tafrov, S.T., Sternglanz, R. and Ramakrishnan, V. (1998) Structure of histone acetyltransferase HAT1: a paradigm for the GCN-5 related N-acetyltransferase superfamily, Cell, 94, 427-438. 29) Perrin, D.D., Dempsey, B. and Serjeant, E.P. (1981) pKa predictions for organic acids and bases. Chapman and Hall, London. 30) Bryantsev, V.S., Diallo, M.S. and Goddard, M.A. (2007) pKa calculations of aliphatic amines, diamines, and aminoamides via density functional theory with a Poisson-Boltzmann continuum solvent model, J.Phys.Chem. A,111, 4422-4430. 31) Grimsley, G.R., Scholtz, J.M. and Pace, C.N. (2009) A summary of the measured pK values of ionizable groups in folded proteins, Protein Science, 18, 247-251. 32) Isom, D.G., Castaneda, C.A., Cannon, B.R., and Garcia-Moreno, B. (2011) Large shifts in pKa values of lysine residues buried inside a protein, Proc.Natl.Acad.Sci.USA, 108, 5260-5265. 33) Harms, M.J., Schlessman, J.L., Chimenti, M.S., Sue, G.R., Damjanovic, A., and Garcia-Moreno, B. (2008) A buried lysine that titrates with a normal pKa: role of conformational flexibility at the protein-water interface as a determinant of pKa values, Protein Science 17, 833-845. 34) Ausio, J. and van Holde, K.E. (1986) Histone hyperacetylation: its effects on nucleosome conformation and stability, Biochemistry 25, 1421-1428. 35) Grunstein, M. (1997) Histone acetylation in chromatin structure and transcription. Nature, 389, 349-352.


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Table I Peptide

Amino Acid Analysis1 S G R K

Mass theory (found)

Vmax2 Kb3 min-1 mM

pKa4 est.

1. Ser-1-NH2/Ser-1-OH5 2. Ser-1-NH2/Ser-1-OP6

0.7 [5.0] 0.5 [5.0]

0.8 1.9 0.8 1.9

887.5 (885.1) 966.4 (965.2)

23.0 18.6

37.0 7.8 204.1 10.3

3. Lys-5-NH2/Ser-1-OH 4. Lys-5-NH2/Ser-1-OP

0.8 [5.0] 0.3 [5.0]

0.9 0.8

2.0 1.9

887.5 966.4

(886.7) (966.2)

24.6 28.6

48.3 8.2 140.8 9.4

5. Lys-8-NH2/Ser-1-OH 6. Lys-8-NH2/Ser-1-OP

0.6 [5.0] 0.4 [5.0]

0.7 0.8

1.7 1.9

887.5 (887.5) 966.4 (968.1)

22.7 23.2

55.6 92.6

8.3 8.8

_______________________________________________________________ 1

The amino acid sequence of all peptides is SGRGKGGKG. When the free amino group at either Ser-1(peptides 1&2), Lys-5 (peptides 3&4), or Lys-8 (5&6) was studied, the other amines were blocked by prior complete acetylation during synthesis. Ser-1 hydroxyl was either phosphorylated or remained free and the C-terminals were amidated. The theoretical amino acid composition is S=1, R=1, K=2, G=5.0. Gly is in brackets since it is the value to which all other amino acids were compared. Serine is partially decomposed during acid hydrolysis prior to analysis so its value is expectedly low. Each peptide had a different pattern of acetylation/phosphorylation during synthesis. 2

Calculated from the y-axis intercept when 1/MAP = 0, Figure 1 panels B,D,F.

3

Calculated from the negative x-axis intercept when 1/Acetylation Rate = 0 ( Figure 1 panels B,D,F.


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Biochemistry

4

These values are estimates calculated from Figure 3 as described in the text.

5

Serine with a free hydroxyl group.

6

Serine with a phosphorylated hydroxyl group.

Figure Legends Figure 1. The effects of phosphorylation of Ser-1 of histone H4 peptides on the acetylation of the amines at Ser-1, Lys-5, and Lys-8. The acetylation rate was calculated from the slope of the plot of the amount of amine remaining after treatment with varying concentrations of MAP for 1 minute time intervals up to 15 minutes. The results were plotted as semi-log plots as % amine remaining over time as shown in Figure 1 of reference (4). Panels A,C, and E are MichaelisMenten type plots. Panels B,D, and F are Lineweaver-Burk type plots where the horizontal red arrows are the reciprocal of Vmax and the vertical red arrows are the reciprocals of Kb. Figure 2- Proposed relationship between Henderson-Hasselbalch and MichaelisMenten equations. This linkage affects acetylation of amines as a function of their basicities (pKa values). Figure 3- Linkage between free amine/protonated amine ratio (left y-axis) vs Kb values (x-axis). The y-axis ratios were calculated from the HendersonHasselbach equation and the x-axis values from the Lineweaver-Burk plots in Figure 1 as described in the text. The line was drawn between the Glyamide and the Gly values (black boxes) since their pKa values are known (see text); their Kb values were calculated as described in ref. (8). The Kb value for each peptide was calculated from Figure 1 ( negative x-axis values of panels B,D,F) and placed on the line. The corresponding left y-axis value was read from the graph and used in the Henderson-Hasselbalch equation to estimate pKa values ( right y-axis).


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

For Table of Contents Only

"Phosphorylation of Serine Induces Lysine pKa Increases in Histone N-terminals and Signaling for Acetylation. Transcription Implications" by Lois R. Manning and James M. Manning


Page 16 of 19

Page 17 of 19

Acetylation of Ser-1 in Histone H4 Peptide

Acetylation of Lys-8 in Histone H4 Peptide

Acetylation of Lys-5 in Histone H4 Peptide

A

24

C

24

E

24

20

20

20

12 8

Ser-1-OP

16

Ser-1-OH

12 Ser-1-OP

8 4

4

0

20

40

60

80

0

100

20

40

60

80

100

.50

20

40

60

80

100

F .50 Ser-1-OP

Ser-1-OP .40

.20

.40 Ser-1-OP

1

.30

.20

Acetylation Rate

1

.30

Acetylation Rate

.40

.30

.20

.10

.10

.10

0

0

0

.08

Ser-1-OH

Ser-1-OH

Ser-1-OH

.04

Ser-1-OP

8

[MAP] (mM)

.50

1 [MAP]

12

0

D

0

Ser-1-OH

[MAP] (mM)

B

-.04

16

4

[MAP] (mM)

1

Acetylation Rate

16

Acetylation Rate

Acetylation Rate

Ser-1-OH

Acetylation Rate

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Biochemistry

-.04

0

.04 1 [MAP]

.08


-.04

0

.04 1 [MAP]

.08

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Page 18 of 19

Henderson-Hasselbalch pH = pKa + log -NH2/-NH3+ (Histone) MAP Ac-N- (Histone) H

[ -NH2/-NH3+ • MAP ]

Michaelis-Menten ACS Paragon Plus Environment

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Biochemistry


Phosphorylation of Serine Induces Lysine pKa Increases in Histone N

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