A summary of the measured pK values of the ionizable groups in folded proteins

Abstract

We tabulated 541 measured pK values reported in the literature for the Asp, Glu, His, Cys, Tyr, and Lys side chains, and the C and N termini of 78 folded proteins. The majority of these values are for the Asp, Glu, and His side chains. The average pK values are Asp 3.5 ± 1.2 (139); Glu 4.2 ± 0.9 (153); His 6.6 ± 1.0 (131); Cys 6.8 ± 2.7 (25); Tyr 10.3 ± 1.2 (20); Lys 10.5 ± 1.1 (35); C-terminus 3.3 ± 0.8 (22) and N-terminus 7.7 ± 0.5 (16). We compare these results with the measured pK values of these groups in alanine pentapeptides, and comment on our overall findings.

Introduction

About 25% of the residues in proteins contain ionizable side chains that are of crucial importance to the mechanism of action of enzymes and to the binding of proteins to other small and large molecules. In addition, the ionization state of these residues determines the net charge on a protein, and this is important to the structure, function, stability, and solubility of a protein. Consequently, biochemists have a strong interest in the pK values of the ionizable groups of proteins and in the factors that perturb them.

We previously published pK values for the ionizable groups of proteins determined with uncharged, alanine pentapeptides.¹ These values reflect the inductive effects of neighboring peptide bonds, but will not be influenced by charge–charge interactions or burial of the ionizable group. Consequently, we think these pKs will serve as reasonable models for the unperturbed pK values of the ionizable groups in proteins. Most of these pKs do not differ significantly from the intrinsic pK values estimated earlier by Nozaki and Tanford.² It will be interesting to compare these values to the pKs determined in unfolded proteins.^3–10

Most of the pK values for folded proteins have been measured directly using techniques based on nuclear magnetic resonance (NMR).^11–13 A smaller number have been measured using indirect techniques.^14,15 In several recent papers, summaries of these results have been given: 212 carboxyl groups¹³; 37 histidine¹⁶; 260 values from 41 proteins¹⁷; 314 values.¹⁸

These previous surveys have provided new insight into the relationship between protein structure and pK values,^13,16 as well as helping those who use theory to predict the pK values of the groups in folded proteins.^17–21 This is interesting because the pK values of many of the ionizable groups in folded proteins are strongly influenced by the local environment.^14,22–29

In this article, we have listed all of the measured pK values that we could find in the literature. The data were taken directly as published from experiments on folded proteins. We summarize our findings and give a few brief comments. We hope this collection of data will be useful to those seeking pK values for proteins, and to those who are interested in how intrinsic pK values can be perturbed in folded proteins.

Results and Discussion

Table I summarizes the measured pK values for 541 ionizable groups from 78 proteins. The individual values, references, and our data analysis are given in the Supporting Information. Data have been collected for 139 Asp, 153 Glu, 131 His, 25 Cys, 20 Tyr, 35 Lys, 22 C-termini, and 16 N-termini. For comparison, the pK values for the same groups measured in uncharged alanine pentapeptides are also given in the table. Here we have corrected the pK for Asp in the pentapeptide from 3.67, as we previously reported ¹ to 3.94. Our new value is based on further measurements under the same conditions on a newly synthesized peptide, where a pK = 3.90 ± 0.02 was measured using a potentiometric titration similar to the technique described in the reference mentioned earlier, and a pK = 3.98 ± 0.05 was determined using NMR (these measurements, as well as others using NMR, will be discussed in an upcoming publication). Therefore, a pK = 3.94 for Asp in the alanine pentapeptide seems most reasonable.

Table I.

Summary of 541 pK Values for 78 Proteins Tabulated from the Literature^a

Group	pK value in alanine pentapeptidesb	Average pK value	Low pK value	High pK value	Number of measurements
Asp	3.9c	3.5 ± 1.2	0.5	9.2	139
Glu	4.3	4.2 ± 0.9	2.1	8.8	153
His	6.5	6.6 ± 1.0	2.4	9.2	131
Cys	8.6	6.8 ± 2.7	2.5	11.1	25
Tyr	9.8	10.3 ± 1.2	6.1	12.1	20
Lys	10.4	10.5 ± 1.1	5.7	12.1	35
C-term	3.7	3.3 ± 0.8	2.4	5.9	22
N-term	8.0	7.7 ± 0.5	6.8	9.1	16

^aThe values were reported under various conditions for 78 folded proteins. The individual measured values can be found in Table 1 of our Supporting Information.

^bFrom Ref. 1.

^cThis value is higher than the one previously reported in the reference above. See text for a discussion of this value.

The majority of the measurements reported in the literature (78%) are for Asp, Glu, and His (Table I and Fig. 1), so most of our comments will be limited to these groups. The average pKs are Asp = 3.5, Glu = 4.2, and His = 6.6. In the case of Asp, the average is 0.4 below the measured value in the alanine pentapeptide (3.5 vs. 3.9). This is clearly reflected by the distribution in Figure 1(A), where ≈77% of the values are lower than the peptide pK of 3.9. In the case of Glu, the average is close to the value measured in the pentapeptide (4.2 vs. 4.3), and 53% of the values are between pH 4–5 [Fig. 1(B)]. The case for His is similar, where the average is again close to the measured value in the alanine pentapeptide (6.6 vs. 6.5). Figure 1(C) shows that ≈44% of the pK measurements fall between pH 6–7, and ≈90% of the His pKs are in the range of pH 5–8.

Figure 1.

Distribution of the measured pK values for Asp (A), Glu (B), and His (C). The arrow on the top axis of each figure indicates the intrinsic pK value as measured in alanine pentapeptides.

The three main factors that will influence the pKs of the ionizable groups in folded proteins are charge–charge interactions, charge–dipole interactions (hydrogen bonding), and the Born effect (dehydration). The pK shift due to the Born effect will depend mainly on the effective dielectric constant of the environment of the ionizable group in the protein compared with the dielectric constant of water (78 at 25°C). The pKs of carboxyl, hydroxyl, and sulfhydryl groups will be increased and the pKs of amino groups decreased in the presence of the lower dielectric constant typically encountered when the groups are partially or completely buried in folded proteins. The presence of a positively charged environment will lower the pKs of all the ionizable groups in a protein, and the presence of a negatively charged environment will raise the pKs. The effect of hydrogen bonding on the pK values will depend on whether the hydrogen bonding is better to the protonated or the deprotonated form of the ionizable group.

The effect of the local environment on the pKs due to the factors mentioned earlier is clearly supported by the low and high values given in Table I. In the case of Asp, the lowest value (0.5) is for Asp70 of T4 lysozyme and for Asp76 of RNase T1. In T4 lysozyme, Asp70 forms a salt bridge with His31, stabilizing the protein by ≈4 kcal mol⁻¹.³⁰ In RNase T1, Asp76 is buried and forms four hydrogen bonds, which account for more than half of the net stability of the protein at pH 7.¹⁴ The highest value (9.2) is Asp 26 in E. coli thioredoxin, where the side chain is completely buried near the active site.^31,32 Another Asp with a pK value near 9 is the V66D variant of staph nuclease, where the side chain is buried in the hydrophobic core of the protein.²⁹ In the case of Glu, the lowest value (2.1) is Glu73 in barnase. This residue participates as a general base at the catalytic site and is close to the positively charged groups of Lys27, Arg83, and Arg 87.³³ The highest value (8.8) is the V66E substitution in staph nuclease that again buries the Glu in the hydrophobic interior of the protein.³⁴ In the case of His, the lowest value (2.4) is for His18 in horse cytochrome c. His18 is coordinated to the iron in the completely buried heme group, and additionally forms two good hydrogen bonds to Ala15 and Pro30³⁵ and this no doubt contributes to its unusually low pK. The highest value (9.2) is for His72 in bovine protein tyrosine phosphatase. Here the imidazole group is buried, but participates in electrostatic interactions with several nearby negatively charged groups.³⁶

There are relatively few pK values reported for Cys (25), Tyr (20), Lys (35), and the N and C termini (16 and 22, respectively). For Cys, the average pK (6.8) is considerably lower than the value measured in alanine pentapeptides (8.6). The Cys residues with the lowest pKs are generally in the active sites of enzymes. For example, the Cys25-S⁻/His159-I-imidazole(Im)⁺ ion pair is found in the active site of cysteine proteinases (see Ref. 18 and Ref. 25 for detailed discussions of these topics). These residues are often studied, and this leads to the low average pK. The lowest value (2.5) is Cys 25 in ficin, a cysteine proteinase, and the highest value (11.1) is the active site Cys in a ubiquitin-conjugating enzyme, Ubc13, where the Cys is surrounded by acidic residues.^37,38

The limited number of measurements for Tyr and Lys is because many proteins unfold in the higher pH range where these groups ionize. The average value for Lys (10.5) does not differ significantly from the pK measured in the pentapeptide (10.4). This is not surprising because the amino group of Lys is usually exposed to solvent in folded proteins (avg % exposed = 66³⁹). The lowest value (5.7) results when a Lys is substituted for Val (V66K) and buried in the hydrophobic core of staph nuclease.⁴⁰ The highest value (12.1) is Lys55 in the Apo-form of calbindin D_9k, where the perturbation is due to the large net negative charge on the protein.⁴¹ For Tyr, the average pK value is 0.5 units above the measured value in the alanine pentapeptide (10.3 vs. 9.8). Again, this is not surprising because the —OH group of Tyr is usually buried in folded proteins (avg % buried = 67³⁹), which would raise the average value.

For the C-termini, the average pK value (3.3) is 0.4 units lower than that measured in our model peptide (3.7). This is not surprising because at low pH where the carboxyl group ionizes, most proteins will have a large positive charge, consequently lowering the pK. The lowest value (2.4) is found in RNase Sa 5K, a basic variant of RNase Sa, with a pI of 10.2.⁴² Furthermore, the C-terminus is a Cys that forms a disulfide bond (Cys96-Cys7).⁴³ This and the large positive charge on the protein are expected to lower the pK significantly.^27,42,44,45 The highest value (5.9) is for Ala79, the C-terminus of subunit c of F₁F₀ ATP Synthase. The high pK results because it is partially buried (see the discussion in Ref. 46). For the N-termini, the average pK value (7.7) does not differ significantly from the pK measured in the peptide (8.0), and the range of low and high values is not as great as that observed for the other groups. The low value (6.8) was measured for the β chain of human deoxyhemoglobin.⁴⁷ The high value (9.1) was measured in RNase Sa, an acidic protein.²⁷ In this case, the large negative charge in the pH range where the N-terminus ionizes undoubtedly contributes to the higher pK.

Methods

The measured pK values for the ionizable groups of Asp, Glu, His, Tyr, Lys, C-termini, and N-termini in 78 proteins were taken from the primary reference given (see Supporting Information for the actual measurements and our data analysis). The technique most used for determining the pK values was NMR, but some measurements were based on less direct techniques. The experiments were performed over a range of temperatures and ionic strength, but in each case, the values were thought to be measured on the folded form of the protein. The significant figures for the measured pK values and the identity of the ionizable groups are given as reported in the original reference. The majority of the data are for wild-type proteins that have been well studied. A few well characterized variants are also included.