histidine pka shifts accompanying the inactivating asp121 asn
TRANSCRIPT
Proc. Natd. Acad. Sci. USA Vol. 88, pp. 8116-8120, September 1991
Biochemistry
Histidine pKa shifts accompanying the inactivating Asp121 Asn substitution in a semisynthetic bovine pancreatic ribonuclease
(NMR/dectrtac efects/Poison-otzman cammlons)
MARK T. CEDERHOLMt, JEANNE A. STUCKEYt, MARILYNN S. DOSCHERt, AND LANA LEEt§ tDepartment of Biochemistry, Wayne State University School of Medicine, Detroit, MI 48201; and tDepartment of Chemistry and Biochemistry, University of Windsor, Windsor, ON N9B 3P4, Canada
Communicated by Frederic M. Richards, June 10, 1991 (received for review March 13, 1991)
ABSTRACT A senisynthetic RNase, RNase-(1-118)-(111- 124), consising of a noncovalent complex between residues 1-118 of RNase (obtained from the proteolytic digestion of RNase A), and a synthetic 14-residue peptide containing resi- dues 111-124 of RNase, exhibits 98% of the enzymatic activity of bovine pancreatic ribonuclease A (EC 3.1.27.5). The re- placement of aspartic acid-121 by asparagine in this semisyn- thetic RNase to form the "D121N" analog reduces kd,/K. to 2.7% of the value for RNase A. In the present work, lH NMR spectroscopy has been used to probe the ionization sates of Pis12, His 9, and His"' in this catalytically defective semisyn- thetic RNase. A comparison of the observed resonances of D121N with those previously determined by others for RNase A enabled us to assign the C2 proton NMR resonances to individual residues; the asignment of Hisll9 was confirmed by titrating D121N with the fully deuterated peptide, [Asnl2l]- RNase-(111-124). The observed pKa values of His'2, HiS"M5, and His"' decrease 0.18, 0.16, and 0.02 pH unit, respectively, as a result of the D121N replacement. Values calculated by using a finite difference algorithm to solve the Poisson- Bodtzmann equation (the DELPH program, version 3.0) and a refined 2.0-A coordinate set for the crystal structure of D121N differ sWficantly for active site residues Hisl2 (ApK. =
-0.58) and Hsl"' (ApK, = -0.55) but not for Hisl"' (ApKa = -0.10). The elimination of bound water from the calulations reduced, but did not reconcile, these discrepancies (His2, ApK. = -0.36; His"9, ApK. = -0.41).
combined with the corresponding peptide in which no amino acid changes have been introduced, the full enzymatic ac- tivity of RNase-(1-118)-(111-124) is generated (6). The over- lap between the peptide and RNase-(1-118), at residues 111-118, is required to achieve both good binding and precise alignment of the two chains (7). A refined crystal structure at 1.8-A resolution of RNase-(1-118)-(111-124), the fully active parent complex, has been determined (8). The assignments of the C2 proton NMR resonances for
each of the four histidines in bovine pancreatic RNase A and their pKa values have been made in several laboratories (9-13). For a review, see ref. 14. The C2 proton NMR spectrum of semisynthetic RNase-(1-118).(111-124) and its pH dependence also have been obtained (15). The titration behavior of the four histidine residues in this semisynthetic derivative was indistinguishable from that found by others for RNase A. We report here the pH titration behavior of the histidine residues in D121N. Using the solution to the Poisson-Boltzmann equation
provided in the electrostatics program DELPHI (16, 17) and the coordinate sets for the crystal structures of both RNase- (1-118) (111-124) (8) and the asparagine analog (18), we have found substantial differences between our experimentally determined values for pKa of D121N minus pKa of RNase A (ApKa) and the ApKa values predicted for the D121N replace- ment.
Several lines of evidence indicate that Asp"21, which is invariant throughout 40 species of mammalian pancreatic RNase (1), functions as part of the active site of bovine pancreatic RNase A (EC 3.1.27.5). Neutron diffraction anal- ysis of single crystals of RNase A has revealed the existence of a hydrogen bond between the carboxyl Q81 of Asp12' and ring N,2 of His119, a critical active site residue (2). The replacement of Asp'21 by asparagine in a semisynthetic derivative of RNase reduces kcat for the small substrate cytidine 2',3'-(cyclic)phosphate at pH 6.0 to 12% of the value for RNase A and increases the value ofKm 4-fold (refs. 3 and 4; M. L. Ram and M.S.D., unpublished data). To delineate further the role of Asp'21 in the function of RNase, we now have determined the apparent pKa values of three of the four histidine residues in the molecule by the measurement of the pH dependence of the C2 proton NMR resonances of the semisynthetic derivative containing the Asn12' replacement. This derivative, "D121N," is prepared by combining RNase- (1-118), a totally inactive entity obtained by successively digesting RNase A with pepsin and carboxypeptidase A (5), with a synthetic peptide composed of the 14 carboxyl- terminal residues of RNase, except that Asp121 has been replaced by asparagine (3, 4). If, instead, RNase-(1-118) is
MATERIALS AND METHODS Materials. RNase A (RAF grade, salt-free, lot 54P6915)
used in the NMR experiments was purchased from Cooper Biomedical. RNase A (type XII-A, lot 13F-8100) used in the preparation of RNase-(1-118) was purchased from Sigma, as were carboxypeptidase A (type I-DFP, lot 13F-8100) and pepsin (P-6887, lot 57F-8105, 4000 units/mg). 2H20,2HCH, NaO2H, and sodium 2,2-dimethyl-2-silapentane-5-sulfonate were purchased from Merck Sharp & Dohme.
Preparation of RNase-(1-118). RNase-(1-118) was pre- pared by the successive digestion ofRNase A with pepsin and carboxypeptidase A (15), except that the gel-filtered prepa- rations were further purified by isocratic ion-exchange chro- matography at 50C on SP-Sephadex G-25 (40- to 120-,um particles; Pharmacia) in 0.13 M sodium phosphate, pH 6.65.
Synthesis of RNase-(111-124) and [Asp'21jRNase-(111-124). RNase-(111-124) and [Asp121]RNase-(111-124) were prepared
Abbreviations: RNase-(1-118), polypeptide consisting of residues 1-118 of RNase A; RNase-(111-124), tetradecapeptide consisting of residues 111-124 of RNase A; [Asp"12]RNase-(111-124), RNase- (111-124) in which Asp'2' has been replaced by asparagine; RNase- (1-118)-(111-124), noncovalent complex of RNase-(1-118) and RNase-(111-124); D121N, noncovalent complex of RNase-(1-118) and RNase-(111-124)(D121N); C2, C2 atom of histidine (39); ApKa, PKa of D121N minus pKa of RNase A, unless otherwise noted; pH*, uncorrected pH of a 2H-containing solution. §To whom reprint requests should be addressed.
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Proc. NatL. Acad. Sci. USA 88 (1991) 8117
by solid-phase synthetic methods (19, 20) and purified by methods previously described (15). NMR Experiments. NMR samples were prepared from
stock solutions of known protein concentration as deter- mined by amino acid analysis. Lyophilized protein derived from aliquot samples of these stock solutions was dissolved in 2H20, adjusted to pH* 3.0 with 1 M 2HC1, and then heated to 60°C for 1 hr to exchange the backbone amide protons (10). The samples were then made 0.3 M in NaCl and 0.5 mM in sodium 2,2-dimethyl-2-silapentane-5-sulfonate and adjusted to the desired pH*. All pH* measurements given are those observed directly and are not corrected for the deuterium isotope effect. pH* measurements were made at room tem- perature with an Ingold 6030-02 microelectrode fitted to a Corning 240 pH meter. The proton NMR spectra were acquired on a Bruker AC-300 NMR spectrometer at 30°C with a spectral width of 4500 Hz, 16,384 data points, and quadrature phase detection. The 1H2HO resonance was re- duced by homonuclear decoupling. The chemical shifts are reported with respect to the principal resonance of sodium 2,2-dimethyl-2-silapentane-5-sulfonate.
Deuteration of [Asp'21JRNase_(111-124). The C2 proton of His"9 in the synthetic peptide [Asp'21]RNase-(111-124) was fully deuterated by incubating a 16.8 mM solution of the peptide at pH* 8.0 at 40°C in 2H20 for 8 days (10). pH Titrations. The pKa values of the C2 protons of RNase
A and the semisynthetic RNase were calculated from a four-parameter nonlinear least-squares curve-fitting program based upon the following function (21, 22):
8obs = 8A + (SAH- 8A) ([Hr)/(Kn + [H]n), where 6A is the chemical shift of the unprotonated species, 8AH is the chemical shift of the protonated species, K is the apparent acid dissociation constant, and n is the Hill coeffi- cient.
Calculation of Electrostatic Potentials. The predicted ApKa values ofthe histidines in the semisynthetic RNase due to the D121N substitution were calculated by the application of a finite difference solution to a combination of the linearized and nonlinearized Poisson-Boltzmann equations using the program DELPHI, version 3.0, on a Silicon Graphics 4D/70GT computer (23, 24). When the refined coordinates ofD121N (2.0-A resolution,
R = 0.187) (18) (Protein Data Bank, reference 2SRN) were used, the N82 of Asn'2' was replaced by an O', and a charge of - /2 was introduced at both 0o1 and O' of this newly introduced aspartic acid; the electrostatic potentials of each of the histidines were then calculated based upon the loss of these two - Y2 charges at this residue. The sulfate anion, which is in the active site in the crystal structures, was not included in the calculations, but all crystallographically bound water molecules were included. The following param- eters were used: ionic strength 0.3 M; protein interior and bound waters, dielectric constant of 2; solvent, dielectric constant of 78.6; grid size, 60 x 60 x 60; focusing boundary conditions and rotational averaging. When the refined coordinates of RNase-(1-118)-(111-124)
(1.8-A resolution, R = 0.204) (8) (Protein Data Bank, refer- ence 1SRN) were used, the effect of the loss of the two -1/2 charges, assumed to be on O81 and O02 of Asp'1 , on the pKa values for the histidines in the protein was calculated. The sulfate anion was again eliminated, and all crystallographi- cally bound water molecules were included. The calculations included the following parameters: ionic strength 0.3 M; protein interior and bound waters, dielectric constant of 2; solvent, dielectric constant of 80; grid size, 65 x 65 x 65; rotational averaging and focusing boundary conditions. For both coordinate sets, the protein solvent boundary was defined by measuring the solvent-accessible surface (25, 26),
using a water probe radius of 1.8 A. One calculation using a probe radius of 1.4 A reduced the ApKa values further by 0.01-0.03 pH unit (see Table 2).
In separate computations, the crystallographically bound waters of the parent complex and the asparagine analog were eliminated and their corresponding ApKa values were calcu- lated.
RESULTS
Histidine 'H NMR Resonances of D121N. Spectrum A of Fig. 1 illustrates the 300-MHz proton NMR resonances ofthe four histidines of native RNase A in 0.3 M NaCl, pH* 4.0, at 300C. This spectrum is in excellent agreement with previously published data obtained under identical conditions (10). These four resonances have previously been assigned to His'2, His"19, His'05, and His' in the order of decreasing chemical shift at pH* 4.0 (9-13). The analogous spectrum for the parent semisynthetic complex, RNase-(1-118)-(111-124), spectrum B in Fig. 1, reveals a direct correspondence with the four histidine resonances found in RNase A (15). How- ever, in this semisynthetic complex, there is a fifth resonance (stippled resonance in spectra B-D of Fig. 1) at 8.6 ppm, which is the same chemical shift as seen in the tripeptide Gly-His-Gly and in RNase-(111-124) (spectrum C) (15). This resonance has been attributed to "unstructured" histidine, which is in slow exchange with those histidines in a native conformation. Spectrum E in Fig. 1 contains the NMR spectrum ofRNase-(1-118); the resonances at 8.88, 8.73, and 8.34 ppm are due to His'2, His105, and His' by analogy with previous studies (15). The additional resonance observed at 8.6 ppm has again been ascribed to "unstructured" histidine; the reason for the broadness of this resonance, with two distinct chemical shifts evident, is not clear. The corresponding spectrum for the asparagine analog,
D121N, in spectrum D in Fig. 1, contains two resonances with chemical shifts previously attributed to His'2 and His48 in both RNase A and RNase-(1-118)-(111-124); these reso- nances, therefore, have been tentatively so assigned in this analog as well.
E
D
9.0 8.8 8.6 8.4 8.2 8.0 8, PPm
FIG. 1. The 300-MHz proton NMR spectra of 2.8 mM RNase A (A), 2.8 mM RNase-(1-118)(111-124) (B), 2.8 mM RNase-(111-124) (C), 2.8 mM D121N (D), and 2.8 mM RNase-(1-118) (E). All samples were at pH* 4.0. Spectra B, C, and D are fully relaxed; spectraA and E are partially relaxed. Shadings are explained in the text. Numbers in spectrum A are histidine positions.
.C
8118 Biochemistry: Cederholm et al.
The resonance due to His"9 was assigned by titrating D121N with a fully deuterated preparation of the tetrade- capeptide [Asp121]RNase-(111-124) at pH* 7.0. In a separate experiment (data not shown), the addition of 1.5 equivalents of the fully deuterated peptide to 2.8 mM D121N at pH* 7.08 caused the resonance at 7.94 ppm, previously attributed to His119, to decrease in intensity whereas those resonances assigned to His12 (7.82 ppm) and His"05 (8.04 ppm) were unchanged, confirming that the resonance at 7.94 ppm is due to His"9. Three ancillary resonances (cross-hatched) appear in the
spectrum of D121N (spectrum D of Fig. 1) that are not observed in the spectrum of RNase A (spectrum A) or in the parent complex (spectrum B), but which do appear in the spectrum of RNase-(1-118) alone (spectrum E). Their pres- ence in the spectrum of D121N suggests that the strength of binding between RNase-(1-118) and RNase-(111-124) may be
A
a
- ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~I
U
-"%...F
J
reduced when asparagine replaces Asp'21; kinetic evidence supports this postulate (see Discussion). These three reso- nances became progressively smaller and disappeared as the pH* was raised from 4.0 to 5.1, so their presence did not interfere significantly with the tracing of the C2 resonances during the pH titrations (see below). Undeuterated [Asp'21]RNase-(111-124) (data not shown) exhibited the same chemical shift as was seen in "unstructured" histidine at 8.6 ppm in RNase-(111-124) (spectrum C in Fig. 1). Histdne Titration Curves. NMR spectra over the range of
7.5-9.0 ppm at selected pH* values are shown for RNase A and D121N in Fig. 2. The resonance of His"9 in the aspar- agine analog has been assigned as discussed above, while His'2 and His'05 have been assigned by comparison with the previously published proton NMR spectra of RNase A. Fig. 3 A andB shows plots of the chemical shifts ofthe C2 protons of His12, His105, and His"19 of RNase A and of D121N, respectively, as a function ofpH. Analysis of the pH titration behavior of His' was not possible due to the broadening of this resonance over the pH range and under the conditions used in these experiments (27), a phenomenon that has been observed previously for RNase A (27, 28) and for RNase- (1-118).(111-124) as well (15). The pKa values and Hill coefficients for the remaining three
accessible histidines, calculated from the nonlinear least- squares analysis described in Materials and Methods, are presented in Table 1. In comparison with the pKa values of RNase A, the pKa values of His12 and His 05 of D121N decrease by 0.18 and 0.16 pH unit, respectively, when asparagine replaces aspartic acid at position 121. In contrast, the corresponding pKa value of His"9 is not altered by this substitution. Modeing the Electrostatic Effect of the ASp'21 -+ Asn
Substitution on the Histidine pK. Values. A refined coordinate set for D121N (18) was used in conjunction with a finite
9.08:8 8'6 8.4 8:2 8.0 i.8 76 8, ppm
E A.
8.8 8.6 84 8.2 8.0 7.8 7.6 8, ppm
FIG. 2. Spectra A-E, 300-MHz proton NMR spectra of 2.8 mM RNase at selected pH* values: A, 4.00; B, 5.04; C, 6.00; D, 7.01; and E, 7.98. Spectra F-J, 2.8 mM D121N at selected pH* values: F, 4.00; G, 5.09; H, 6.03; I, 7.07; and J, 7.99. The C2 protons of His'2 (U), His'05 (e), and His"19 (A) have been labeled. Both samples were in 0.3 M NaCl at 30°C in 2H20.
8.5 E
ce 8.0
7.5 3.5
7.5 . .
3.5 4.5 5.5 6.5 7.5 8.5 pH
FIG. 3. Chemical shifts of the C2 protons of His12 (-), His'05 (-), and His"9 (A) of RNase A (A) and D121N (B) as a function of pH. Both samples were 0.3 M NaCl at 30°C in 2H20. The solid lines represent the calculated chemical shifts determined by the nonlinear least-squares analysis described in Materials and Methods.
Proc. NaM Acad Sci. USA 88 (1991)
Proc. Natl. Acad. Sci. USA 88 (1991) 8119
Table 1. Least-squares analysis of histidine titration profiles Residue* System PA6 pKa n His'2 RNase-(1-118).(111-124)t 7.67 (0.01) 9.02 (0.01) 5.94 (0.02) 0.69 (0.02)
RNase At 7.64 (0.03) 8.96 (0.03) 6.03 (0.02) 0.74 (0.01) D121Nt 7.70 (0.02) 8.97 (0.01) 5.85 (0.01) 0.79 (0.01)
His'05 RNase-(1-118).(111-124)t 7.69 (0.02) 8.76 (0.01) 6.78 (0.02) 0.89 (0.03) RNase At 7.69 (0.01) 8.75 (0.02) 6.82 (0.01) 0.94 (0.01) D121Nt 7.70 (0.02) 8.78 (0.02) 6.66 (0.01) 0.88 (0.01)
His"19 RNase-(1-118)-(111-124)t 7.76 (0.01) 8.83 (0.01) 6.26 (0.02) 0.77 (0.03) RNase At 7.76 (0.02) 8.80 (0.02) 6.33 (0.01) 0.86 (0.01) D121N§ 7.75 (0.02) 8.77 (0.01) 6.31 (0.01) 0.87 (0.01)
Parameters are based on ref. 21. Numbers in parentheses represent standard deviations from the least-squares fits. *Assignments based on refs. 9-13. tValues from published measurements (15). tPutative assignment based on correlation of chemical shifts with RNase A; values from measurements of spectra shown in Fig. 2. §Assignment made by deuteration (see Results).
difference solution to the Poisson-Boltzmann equation (DEL- PHI, version 3.0) (23, 24) to calculate the expected changes in the pKa values ofthe three histidine residues as a result ofthe asparagine substitution. The observed ApKa value of -0.16 for His'05 is in good agreement with the predicted value of -0.10 for this residue (Table 2, rows 1 and 4). In contrast, the predicted ApKa values for His'2 and His'19 of -0.58 and -0.55, respectively, are significantly greater than those of -0.18 and -0.02 found experimentally (Table 2, rows 1 and 4).
If the numerous small structural changes with respect to protein and crystallographically bound water that accompany the substitution of asparagine for Asp12' (18) are ignored by calculating electrostatic potentials using the coordinate set for RNase-(1-118) (111-124) (8), the discrepancy between the experimental and the predicted ApKa values for His12 and His"9 resulting from the loss of two -1/2 charges at o01 and O02 of Asp12' is still greater (Table 2, rows 1 and 3). Again, the agreement between the experimental (-0.16) and pre- dicted (-0.15) pKa shift for His'05 is excellent. When crystallographically bound water molecules were
eliminated in the ApKa calculations, all of the values pre- dicted using the RNase-(1-118)-(111-124) coordinate set (Ta- ble 2, row 5) or the D121N coordinate set (Table 2, row 6) decreased in magnitude. Significant discrepancies between the experimental and theoretical values, nevertheless, re- main.
Table 2. Comparison of the observed and predicted histidine PKa changes in the semisynthetic RNases
ApKa, pH units
Row His'2 His'05 His"19 Comments 1* -0.18 -0.16 -0.02 2t -0.09 -0.12 +0.05 3* -0.85 -0.15 -1.1 Asp'2'§, +H201 4O -0.58 -0.10 -0.55 Asn'21i, +H20¶ 51t -0.39 -0.09 -0.41 Asp'21§, -H201 6t -0.36 -0.07 -0.41 Asnl2l**, -H2O0** *Experimentally determined pKa of D121N minus pKa of RNase A. tExperimentally determined pKa of D121N minus pKa of RNase- (1-118)-(111-124) (15). fCalculated by computer simulation (DELPHI, version 3.0) as de- scribed in Materials and Methods. §Based upon the coordinates of RNase-(1-118)-(111-124) (8). fThe + and - signs indicate the presence and absence of crystallo- graphically bound water. I"Based upon the coordinates of D121N (18). **Use of a 1.4A water probe radius provided ApKa values of -0.35,
-0.06, and -0.38 for His'2, His'05, and His"19, respectively.
DISCUSSION The similarity of the chemical shifts of fully protonated and fully deprotonated His'2, His105, and His"19 in RNase A, RNase-(1-118).(111-124), and D121N listed in Table 1 sug- gests that the environments of these three histidine residues are similar in all three molecules at very low pH and at very high pH. Even at pH* 4.0, the proton NMR spectrum of D121N contains resonances that correspond well with those ofHis'2 and His' in the parent complex and in RNase A (Fig. 1). At this pH* value, however, the chemical shifts of His'05 and His"19 in the asparagine analog are significantly different. The substitution of asparagine at position 121 has also resulted in a decrease in the observed pKa values ofHis12 and His'05 of 0.18 and 0.16 pH unit, respectively (Table 1). Thus, the environments of these three histidine residues at inter- mediate pH values have evidently all been disturbed by this mutation. Some decrease in the pKa values of the histidine residues
was anticipated, as the replacement of aspartic acid by asparagine removes a negative charge from the molecule; this change would be expected to destabilize the positively charged protonated form of a histidine residue and concom- itantly decrease its pKa value. Such an electrostatic effect is sharply dependent upon distance and ionic strength, but it has been shown experimentally to remain detectable at considerable distances and substantial ionic strengths. In an extracellular subtilisin from Bacillus amyloliquefaciens, Rus- sell and coworkers (29, 30) have observed that the replace- ment of Asp" with serine reduced the pKa of the active site His' by 0.29 pH unit at an ionic strength of 0.1 M (ApKa = -0.29). These residues are separated by 12-13 A. At an ionic strength of 0.5 M, a corresponding decrease of 0.10 pH unit (APKa = -0.10) could still be detected. The calculation ofthe electrostatic potentials in this subtilisin by the finite differ- ence Poisson-Boltzmann method (17) resulted in excellent agreement between the experimentally determined and pre- dicted ApKa values for His' (23). In a second example, a dramatic decrease of 1.5 pH units occurs in the pKa of His57 in bovine pancreatic trypsin when Asp102, to which His57 is hydrogen bonded, is replaced by an asparagine (31). No major structural rearrangements result from this substitution (32).
In RNase A, His"9 is found in a conformation that brings the side chains of His"9 (NW2) and Asp'12 (Q81) within hydrogen bonding distance (2.74 A) (33-35), whereas in both RNase-(1-118)-(111-124) and the asparagine analog, His"19 occupies predominantly a second conformation that is achieved by rotation around the Ca-C0 bond (8, 36). In this conformation, the distance between these two residues is considerably greater (9.9 and 8.8 A, respectively) (8, 18).
Biochemistry: Cederholm et al.
8120 Biochemistry: Cederholm et al.
Regardless of its positioning, however, a sizeable decrease in the pK. value for His119 was expected, and, indeed, the results from the application of the Poisson-Boltzmann equa- tion confirmed this expectation. In addition, these calcula- tions have revealed that the pK. shift for His12 is also substantially muted. The discrepancies between the observed and predicted
pK5 values for His12 and His119 may be the result of a number of factors. First, we have used the coordinates for crystal structures in 3 M ammonium sulfate to model the titration behavior of a protein in solution in 0.3 M NaCl. Second, with regard to His 19, the asparagine substitution has resulted in the imidazole ring ofthis residue undergoing a 1800 flip so that the N81 of the ring now forms a strong hydrogen bond to a water molecule (18). A third factor may be the effect of changes in the arrangement of bound water molecules. A comparison of the structures of RNase-(1-118)*(111-124) and D121N reveals numerous differences in the location and structure of crystallographically bound water networks (18). Such rearrangements may have resulted in significant changes in local dielectric constant. For RNase-(1-118).(111- 124) and D121N, the initial ApKa calculations included crys- tallographically bound water molecules, which were assigned a conventional dielectric constant of 2 (37). In both cases, large differences between the experimental (Table 2, rows 1 and 2) and theoretical (Table 2, rows 3 and 4) ApKa values were observed. However, when the crystallographically bound water molecules of the parent complex and- of the asparagine derivative were eliminated, there was a reduction in these discrepancies (Table 2, rows 5 and 6). This obser- vation suggests that the dielectric constant of bound water molecules may be closer to that of bulk solvent. In the case of lysozyme, better results were also obtained after removal ofbound water molecules from the crystallographic structure (38). When the coordinates for D121N are used in the presence
of crystallographically bound water, the discrepancy be- tween the observed and the predicted ApK. values (Table 2, rows 1 and 4) is moderated compared with the values obtained by using the coordinate set for the parent complex (Table 2, rows 1 and 3). This moderation suggests that the structure of the protein as a whole is accommodating (or attempting to accommodate) to the change in charge distri- bution resulting from the asparagine substitution. Such an accommodation results, therefore, in a multitude of small, but significant changes in structure throughout the molecule (18). Three ancillary resonances are seen in the proton NMR
spectra of D121N over the pH* range of 4.0-5.1 that are not seen with RNase A or with the parent complex; they do appear in the spectrum of free RNase-(1-118X, however (Fig. 1, spectrum E). The reduced binding energy between the asparagine-containing peptide and RNase-(1-118) indicated by this observation is supported by kinetic measurements at pH 6.0: Kd = 33 ,uM (vs. 1 FxM for the parent complex) (M. L. Ram and M.S.D., unpublished data). It is not likely that the presence of significant amounts of free RNase-(1-118) and [Asp121]RNase-(111-124) in the pH range 4.0-5.1 has seri- ously perturbed the titration curves of the histidine residues in the asparagine analog. If the pKa of the controlling group is 4.0 (as indicated by the presence of essentially equal concentrations of the complex and its two components at this pH value), only 7% of the chains remain dissociated at pH 5.1. Moreover, at pH 5.1, only 6% of His119 (pKa = 6.3) and 17% of His12 (pKa = 5.8) will have been titrated. The increase in Km and the reduction in kcat that accom-
pany the replacement of Asp121 by asparagine in RNase result in an enzyme that exhibits 6% activity against cytidine 2',3'-(cyclic)phosphate at pH 6.0 under standard assay con- ditions (refs. 3 and 4; M. L. Ram and M.S.D., unpublished
data). The present study has eliminated models in which this inactivation is associated with a drastic decrease in the ground state pKa value of active site His12 or His119, a distinct possibility a priori. Further measurements in the presence of active-site ligands may reveal differences in pKa values that would help to clarify the basis for the inactivation.
We thank Dr. Brian F. Pi Edwards, Dr. Philip D. Martin, and Dr. V. Srini J. deMel for providing the coordinates ofD121N. Amino acid analyses were performed by the Wayne State University Macromo- lecular Core Facility (supported in part by the Wayne State Univer- sity Center for Molecular Biology). This work was supported in part by the National Science and Engineering Research Council of Canada, the J. P. Bickell Foundation, The University of Windsor Research Board, and National Institutes of Health GrantGM 40630.
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327-335. 17. Sharp, K. A. & Honig, B. (1990) Annu. Rev. Biophys. Biophys. Chem.
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Proteins 1, 47-59. 24. Gilson, M. K. & Honig, B. H. (1987) Nature (London) 330, 84-86. 25. Lee, B. & Richards, F. M. (1971) J. Mol. Biol. 55, 379-400. 26. Richards, F. M. (1985) Methods Enzymol. 115, 440-464. 27. Roberts, G. C. K., Meadows, D. H.&Jardetzky,O. (1969) Biochemistry
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Proc. Natl. Acad. Sci. USA 88 (1991)
Histidine pKa shifts accompanying the inactivating Asp121 Asn substitution in a semisynthetic bovine pancreatic ribonuclease
(NMR/dectrtac efects/Poison-otzman cammlons)
MARK T. CEDERHOLMt, JEANNE A. STUCKEYt, MARILYNN S. DOSCHERt, AND LANA LEEt§ tDepartment of Biochemistry, Wayne State University School of Medicine, Detroit, MI 48201; and tDepartment of Chemistry and Biochemistry, University of Windsor, Windsor, ON N9B 3P4, Canada
Communicated by Frederic M. Richards, June 10, 1991 (received for review March 13, 1991)
ABSTRACT A senisynthetic RNase, RNase-(1-118)-(111- 124), consising of a noncovalent complex between residues 1-118 of RNase (obtained from the proteolytic digestion of RNase A), and a synthetic 14-residue peptide containing resi- dues 111-124 of RNase, exhibits 98% of the enzymatic activity of bovine pancreatic ribonuclease A (EC 3.1.27.5). The re- placement of aspartic acid-121 by asparagine in this semisyn- thetic RNase to form the "D121N" analog reduces kd,/K. to 2.7% of the value for RNase A. In the present work, lH NMR spectroscopy has been used to probe the ionization sates of Pis12, His 9, and His"' in this catalytically defective semisyn- thetic RNase. A comparison of the observed resonances of D121N with those previously determined by others for RNase A enabled us to assign the C2 proton NMR resonances to individual residues; the asignment of Hisll9 was confirmed by titrating D121N with the fully deuterated peptide, [Asnl2l]- RNase-(111-124). The observed pKa values of His'2, HiS"M5, and His"' decrease 0.18, 0.16, and 0.02 pH unit, respectively, as a result of the D121N replacement. Values calculated by using a finite difference algorithm to solve the Poisson- Bodtzmann equation (the DELPH program, version 3.0) and a refined 2.0-A coordinate set for the crystal structure of D121N differ sWficantly for active site residues Hisl2 (ApK. =
-0.58) and Hsl"' (ApK, = -0.55) but not for Hisl"' (ApKa = -0.10). The elimination of bound water from the calulations reduced, but did not reconcile, these discrepancies (His2, ApK. = -0.36; His"9, ApK. = -0.41).
combined with the corresponding peptide in which no amino acid changes have been introduced, the full enzymatic ac- tivity of RNase-(1-118)-(111-124) is generated (6). The over- lap between the peptide and RNase-(1-118), at residues 111-118, is required to achieve both good binding and precise alignment of the two chains (7). A refined crystal structure at 1.8-A resolution of RNase-(1-118)-(111-124), the fully active parent complex, has been determined (8). The assignments of the C2 proton NMR resonances for
each of the four histidines in bovine pancreatic RNase A and their pKa values have been made in several laboratories (9-13). For a review, see ref. 14. The C2 proton NMR spectrum of semisynthetic RNase-(1-118).(111-124) and its pH dependence also have been obtained (15). The titration behavior of the four histidine residues in this semisynthetic derivative was indistinguishable from that found by others for RNase A. We report here the pH titration behavior of the histidine residues in D121N. Using the solution to the Poisson-Boltzmann equation
provided in the electrostatics program DELPHI (16, 17) and the coordinate sets for the crystal structures of both RNase- (1-118) (111-124) (8) and the asparagine analog (18), we have found substantial differences between our experimentally determined values for pKa of D121N minus pKa of RNase A (ApKa) and the ApKa values predicted for the D121N replace- ment.
Several lines of evidence indicate that Asp"21, which is invariant throughout 40 species of mammalian pancreatic RNase (1), functions as part of the active site of bovine pancreatic RNase A (EC 3.1.27.5). Neutron diffraction anal- ysis of single crystals of RNase A has revealed the existence of a hydrogen bond between the carboxyl Q81 of Asp12' and ring N,2 of His119, a critical active site residue (2). The replacement of Asp'21 by asparagine in a semisynthetic derivative of RNase reduces kcat for the small substrate cytidine 2',3'-(cyclic)phosphate at pH 6.0 to 12% of the value for RNase A and increases the value ofKm 4-fold (refs. 3 and 4; M. L. Ram and M.S.D., unpublished data). To delineate further the role of Asp'21 in the function of RNase, we now have determined the apparent pKa values of three of the four histidine residues in the molecule by the measurement of the pH dependence of the C2 proton NMR resonances of the semisynthetic derivative containing the Asn12' replacement. This derivative, "D121N," is prepared by combining RNase- (1-118), a totally inactive entity obtained by successively digesting RNase A with pepsin and carboxypeptidase A (5), with a synthetic peptide composed of the 14 carboxyl- terminal residues of RNase, except that Asp121 has been replaced by asparagine (3, 4). If, instead, RNase-(1-118) is
MATERIALS AND METHODS Materials. RNase A (RAF grade, salt-free, lot 54P6915)
used in the NMR experiments was purchased from Cooper Biomedical. RNase A (type XII-A, lot 13F-8100) used in the preparation of RNase-(1-118) was purchased from Sigma, as were carboxypeptidase A (type I-DFP, lot 13F-8100) and pepsin (P-6887, lot 57F-8105, 4000 units/mg). 2H20,2HCH, NaO2H, and sodium 2,2-dimethyl-2-silapentane-5-sulfonate were purchased from Merck Sharp & Dohme.
Preparation of RNase-(1-118). RNase-(1-118) was pre- pared by the successive digestion ofRNase A with pepsin and carboxypeptidase A (15), except that the gel-filtered prepa- rations were further purified by isocratic ion-exchange chro- matography at 50C on SP-Sephadex G-25 (40- to 120-,um particles; Pharmacia) in 0.13 M sodium phosphate, pH 6.65.
Synthesis of RNase-(111-124) and [Asp'21jRNase-(111-124). RNase-(111-124) and [Asp121]RNase-(111-124) were prepared
Abbreviations: RNase-(1-118), polypeptide consisting of residues 1-118 of RNase A; RNase-(111-124), tetradecapeptide consisting of residues 111-124 of RNase A; [Asp"12]RNase-(111-124), RNase- (111-124) in which Asp'2' has been replaced by asparagine; RNase- (1-118)-(111-124), noncovalent complex of RNase-(1-118) and RNase-(111-124); D121N, noncovalent complex of RNase-(1-118) and RNase-(111-124)(D121N); C2, C2 atom of histidine (39); ApKa, PKa of D121N minus pKa of RNase A, unless otherwise noted; pH*, uncorrected pH of a 2H-containing solution. §To whom reprint requests should be addressed.
8116
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Proc. NatL. Acad. Sci. USA 88 (1991) 8117
by solid-phase synthetic methods (19, 20) and purified by methods previously described (15). NMR Experiments. NMR samples were prepared from
stock solutions of known protein concentration as deter- mined by amino acid analysis. Lyophilized protein derived from aliquot samples of these stock solutions was dissolved in 2H20, adjusted to pH* 3.0 with 1 M 2HC1, and then heated to 60°C for 1 hr to exchange the backbone amide protons (10). The samples were then made 0.3 M in NaCl and 0.5 mM in sodium 2,2-dimethyl-2-silapentane-5-sulfonate and adjusted to the desired pH*. All pH* measurements given are those observed directly and are not corrected for the deuterium isotope effect. pH* measurements were made at room tem- perature with an Ingold 6030-02 microelectrode fitted to a Corning 240 pH meter. The proton NMR spectra were acquired on a Bruker AC-300 NMR spectrometer at 30°C with a spectral width of 4500 Hz, 16,384 data points, and quadrature phase detection. The 1H2HO resonance was re- duced by homonuclear decoupling. The chemical shifts are reported with respect to the principal resonance of sodium 2,2-dimethyl-2-silapentane-5-sulfonate.
Deuteration of [Asp'21JRNase_(111-124). The C2 proton of His"9 in the synthetic peptide [Asp'21]RNase-(111-124) was fully deuterated by incubating a 16.8 mM solution of the peptide at pH* 8.0 at 40°C in 2H20 for 8 days (10). pH Titrations. The pKa values of the C2 protons of RNase
A and the semisynthetic RNase were calculated from a four-parameter nonlinear least-squares curve-fitting program based upon the following function (21, 22):
8obs = 8A + (SAH- 8A) ([Hr)/(Kn + [H]n), where 6A is the chemical shift of the unprotonated species, 8AH is the chemical shift of the protonated species, K is the apparent acid dissociation constant, and n is the Hill coeffi- cient.
Calculation of Electrostatic Potentials. The predicted ApKa values ofthe histidines in the semisynthetic RNase due to the D121N substitution were calculated by the application of a finite difference solution to a combination of the linearized and nonlinearized Poisson-Boltzmann equations using the program DELPHI, version 3.0, on a Silicon Graphics 4D/70GT computer (23, 24). When the refined coordinates ofD121N (2.0-A resolution,
R = 0.187) (18) (Protein Data Bank, reference 2SRN) were used, the N82 of Asn'2' was replaced by an O', and a charge of - /2 was introduced at both 0o1 and O' of this newly introduced aspartic acid; the electrostatic potentials of each of the histidines were then calculated based upon the loss of these two - Y2 charges at this residue. The sulfate anion, which is in the active site in the crystal structures, was not included in the calculations, but all crystallographically bound water molecules were included. The following param- eters were used: ionic strength 0.3 M; protein interior and bound waters, dielectric constant of 2; solvent, dielectric constant of 78.6; grid size, 60 x 60 x 60; focusing boundary conditions and rotational averaging. When the refined coordinates of RNase-(1-118)-(111-124)
(1.8-A resolution, R = 0.204) (8) (Protein Data Bank, refer- ence 1SRN) were used, the effect of the loss of the two -1/2 charges, assumed to be on O81 and O02 of Asp'1 , on the pKa values for the histidines in the protein was calculated. The sulfate anion was again eliminated, and all crystallographi- cally bound water molecules were included. The calculations included the following parameters: ionic strength 0.3 M; protein interior and bound waters, dielectric constant of 2; solvent, dielectric constant of 80; grid size, 65 x 65 x 65; rotational averaging and focusing boundary conditions. For both coordinate sets, the protein solvent boundary was defined by measuring the solvent-accessible surface (25, 26),
using a water probe radius of 1.8 A. One calculation using a probe radius of 1.4 A reduced the ApKa values further by 0.01-0.03 pH unit (see Table 2).
In separate computations, the crystallographically bound waters of the parent complex and the asparagine analog were eliminated and their corresponding ApKa values were calcu- lated.
RESULTS
Histidine 'H NMR Resonances of D121N. Spectrum A of Fig. 1 illustrates the 300-MHz proton NMR resonances ofthe four histidines of native RNase A in 0.3 M NaCl, pH* 4.0, at 300C. This spectrum is in excellent agreement with previously published data obtained under identical conditions (10). These four resonances have previously been assigned to His'2, His"19, His'05, and His' in the order of decreasing chemical shift at pH* 4.0 (9-13). The analogous spectrum for the parent semisynthetic complex, RNase-(1-118)-(111-124), spectrum B in Fig. 1, reveals a direct correspondence with the four histidine resonances found in RNase A (15). How- ever, in this semisynthetic complex, there is a fifth resonance (stippled resonance in spectra B-D of Fig. 1) at 8.6 ppm, which is the same chemical shift as seen in the tripeptide Gly-His-Gly and in RNase-(111-124) (spectrum C) (15). This resonance has been attributed to "unstructured" histidine, which is in slow exchange with those histidines in a native conformation. Spectrum E in Fig. 1 contains the NMR spectrum ofRNase-(1-118); the resonances at 8.88, 8.73, and 8.34 ppm are due to His'2, His105, and His' by analogy with previous studies (15). The additional resonance observed at 8.6 ppm has again been ascribed to "unstructured" histidine; the reason for the broadness of this resonance, with two distinct chemical shifts evident, is not clear. The corresponding spectrum for the asparagine analog,
D121N, in spectrum D in Fig. 1, contains two resonances with chemical shifts previously attributed to His'2 and His48 in both RNase A and RNase-(1-118)-(111-124); these reso- nances, therefore, have been tentatively so assigned in this analog as well.
E
D
9.0 8.8 8.6 8.4 8.2 8.0 8, PPm
FIG. 1. The 300-MHz proton NMR spectra of 2.8 mM RNase A (A), 2.8 mM RNase-(1-118)(111-124) (B), 2.8 mM RNase-(111-124) (C), 2.8 mM D121N (D), and 2.8 mM RNase-(1-118) (E). All samples were at pH* 4.0. Spectra B, C, and D are fully relaxed; spectraA and E are partially relaxed. Shadings are explained in the text. Numbers in spectrum A are histidine positions.
.C
8118 Biochemistry: Cederholm et al.
The resonance due to His"9 was assigned by titrating D121N with a fully deuterated preparation of the tetrade- capeptide [Asp121]RNase-(111-124) at pH* 7.0. In a separate experiment (data not shown), the addition of 1.5 equivalents of the fully deuterated peptide to 2.8 mM D121N at pH* 7.08 caused the resonance at 7.94 ppm, previously attributed to His119, to decrease in intensity whereas those resonances assigned to His12 (7.82 ppm) and His"05 (8.04 ppm) were unchanged, confirming that the resonance at 7.94 ppm is due to His"9. Three ancillary resonances (cross-hatched) appear in the
spectrum of D121N (spectrum D of Fig. 1) that are not observed in the spectrum of RNase A (spectrum A) or in the parent complex (spectrum B), but which do appear in the spectrum of RNase-(1-118) alone (spectrum E). Their pres- ence in the spectrum of D121N suggests that the strength of binding between RNase-(1-118) and RNase-(111-124) may be
A
a
- ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~I
U
-"%...F
J
reduced when asparagine replaces Asp'21; kinetic evidence supports this postulate (see Discussion). These three reso- nances became progressively smaller and disappeared as the pH* was raised from 4.0 to 5.1, so their presence did not interfere significantly with the tracing of the C2 resonances during the pH titrations (see below). Undeuterated [Asp'21]RNase-(111-124) (data not shown) exhibited the same chemical shift as was seen in "unstructured" histidine at 8.6 ppm in RNase-(111-124) (spectrum C in Fig. 1). Histdne Titration Curves. NMR spectra over the range of
7.5-9.0 ppm at selected pH* values are shown for RNase A and D121N in Fig. 2. The resonance of His"9 in the aspar- agine analog has been assigned as discussed above, while His'2 and His'05 have been assigned by comparison with the previously published proton NMR spectra of RNase A. Fig. 3 A andB shows plots of the chemical shifts ofthe C2 protons of His12, His105, and His"19 of RNase A and of D121N, respectively, as a function ofpH. Analysis of the pH titration behavior of His' was not possible due to the broadening of this resonance over the pH range and under the conditions used in these experiments (27), a phenomenon that has been observed previously for RNase A (27, 28) and for RNase- (1-118).(111-124) as well (15). The pKa values and Hill coefficients for the remaining three
accessible histidines, calculated from the nonlinear least- squares analysis described in Materials and Methods, are presented in Table 1. In comparison with the pKa values of RNase A, the pKa values of His12 and His 05 of D121N decrease by 0.18 and 0.16 pH unit, respectively, when asparagine replaces aspartic acid at position 121. In contrast, the corresponding pKa value of His"9 is not altered by this substitution. Modeing the Electrostatic Effect of the ASp'21 -+ Asn
Substitution on the Histidine pK. Values. A refined coordinate set for D121N (18) was used in conjunction with a finite
9.08:8 8'6 8.4 8:2 8.0 i.8 76 8, ppm
E A.
8.8 8.6 84 8.2 8.0 7.8 7.6 8, ppm
FIG. 2. Spectra A-E, 300-MHz proton NMR spectra of 2.8 mM RNase at selected pH* values: A, 4.00; B, 5.04; C, 6.00; D, 7.01; and E, 7.98. Spectra F-J, 2.8 mM D121N at selected pH* values: F, 4.00; G, 5.09; H, 6.03; I, 7.07; and J, 7.99. The C2 protons of His'2 (U), His'05 (e), and His"19 (A) have been labeled. Both samples were in 0.3 M NaCl at 30°C in 2H20.
8.5 E
ce 8.0
7.5 3.5
7.5 . .
3.5 4.5 5.5 6.5 7.5 8.5 pH
FIG. 3. Chemical shifts of the C2 protons of His12 (-), His'05 (-), and His"9 (A) of RNase A (A) and D121N (B) as a function of pH. Both samples were 0.3 M NaCl at 30°C in 2H20. The solid lines represent the calculated chemical shifts determined by the nonlinear least-squares analysis described in Materials and Methods.
Proc. NaM Acad Sci. USA 88 (1991)
Proc. Natl. Acad. Sci. USA 88 (1991) 8119
Table 1. Least-squares analysis of histidine titration profiles Residue* System PA6 pKa n His'2 RNase-(1-118).(111-124)t 7.67 (0.01) 9.02 (0.01) 5.94 (0.02) 0.69 (0.02)
RNase At 7.64 (0.03) 8.96 (0.03) 6.03 (0.02) 0.74 (0.01) D121Nt 7.70 (0.02) 8.97 (0.01) 5.85 (0.01) 0.79 (0.01)
His'05 RNase-(1-118).(111-124)t 7.69 (0.02) 8.76 (0.01) 6.78 (0.02) 0.89 (0.03) RNase At 7.69 (0.01) 8.75 (0.02) 6.82 (0.01) 0.94 (0.01) D121Nt 7.70 (0.02) 8.78 (0.02) 6.66 (0.01) 0.88 (0.01)
His"19 RNase-(1-118)-(111-124)t 7.76 (0.01) 8.83 (0.01) 6.26 (0.02) 0.77 (0.03) RNase At 7.76 (0.02) 8.80 (0.02) 6.33 (0.01) 0.86 (0.01) D121N§ 7.75 (0.02) 8.77 (0.01) 6.31 (0.01) 0.87 (0.01)
Parameters are based on ref. 21. Numbers in parentheses represent standard deviations from the least-squares fits. *Assignments based on refs. 9-13. tValues from published measurements (15). tPutative assignment based on correlation of chemical shifts with RNase A; values from measurements of spectra shown in Fig. 2. §Assignment made by deuteration (see Results).
difference solution to the Poisson-Boltzmann equation (DEL- PHI, version 3.0) (23, 24) to calculate the expected changes in the pKa values ofthe three histidine residues as a result ofthe asparagine substitution. The observed ApKa value of -0.16 for His'05 is in good agreement with the predicted value of -0.10 for this residue (Table 2, rows 1 and 4). In contrast, the predicted ApKa values for His'2 and His'19 of -0.58 and -0.55, respectively, are significantly greater than those of -0.18 and -0.02 found experimentally (Table 2, rows 1 and 4).
If the numerous small structural changes with respect to protein and crystallographically bound water that accompany the substitution of asparagine for Asp12' (18) are ignored by calculating electrostatic potentials using the coordinate set for RNase-(1-118) (111-124) (8), the discrepancy between the experimental and the predicted ApKa values for His12 and His"9 resulting from the loss of two -1/2 charges at o01 and O02 of Asp12' is still greater (Table 2, rows 1 and 3). Again, the agreement between the experimental (-0.16) and pre- dicted (-0.15) pKa shift for His'05 is excellent. When crystallographically bound water molecules were
eliminated in the ApKa calculations, all of the values pre- dicted using the RNase-(1-118)-(111-124) coordinate set (Ta- ble 2, row 5) or the D121N coordinate set (Table 2, row 6) decreased in magnitude. Significant discrepancies between the experimental and theoretical values, nevertheless, re- main.
Table 2. Comparison of the observed and predicted histidine PKa changes in the semisynthetic RNases
ApKa, pH units
Row His'2 His'05 His"19 Comments 1* -0.18 -0.16 -0.02 2t -0.09 -0.12 +0.05 3* -0.85 -0.15 -1.1 Asp'2'§, +H201 4O -0.58 -0.10 -0.55 Asn'21i, +H20¶ 51t -0.39 -0.09 -0.41 Asp'21§, -H201 6t -0.36 -0.07 -0.41 Asnl2l**, -H2O0** *Experimentally determined pKa of D121N minus pKa of RNase A. tExperimentally determined pKa of D121N minus pKa of RNase- (1-118)-(111-124) (15). fCalculated by computer simulation (DELPHI, version 3.0) as de- scribed in Materials and Methods. §Based upon the coordinates of RNase-(1-118)-(111-124) (8). fThe + and - signs indicate the presence and absence of crystallo- graphically bound water. I"Based upon the coordinates of D121N (18). **Use of a 1.4A water probe radius provided ApKa values of -0.35,
-0.06, and -0.38 for His'2, His'05, and His"19, respectively.
DISCUSSION The similarity of the chemical shifts of fully protonated and fully deprotonated His'2, His105, and His"19 in RNase A, RNase-(1-118).(111-124), and D121N listed in Table 1 sug- gests that the environments of these three histidine residues are similar in all three molecules at very low pH and at very high pH. Even at pH* 4.0, the proton NMR spectrum of D121N contains resonances that correspond well with those ofHis'2 and His' in the parent complex and in RNase A (Fig. 1). At this pH* value, however, the chemical shifts of His'05 and His"19 in the asparagine analog are significantly different. The substitution of asparagine at position 121 has also resulted in a decrease in the observed pKa values ofHis12 and His'05 of 0.18 and 0.16 pH unit, respectively (Table 1). Thus, the environments of these three histidine residues at inter- mediate pH values have evidently all been disturbed by this mutation. Some decrease in the pKa values of the histidine residues
was anticipated, as the replacement of aspartic acid by asparagine removes a negative charge from the molecule; this change would be expected to destabilize the positively charged protonated form of a histidine residue and concom- itantly decrease its pKa value. Such an electrostatic effect is sharply dependent upon distance and ionic strength, but it has been shown experimentally to remain detectable at considerable distances and substantial ionic strengths. In an extracellular subtilisin from Bacillus amyloliquefaciens, Rus- sell and coworkers (29, 30) have observed that the replace- ment of Asp" with serine reduced the pKa of the active site His' by 0.29 pH unit at an ionic strength of 0.1 M (ApKa = -0.29). These residues are separated by 12-13 A. At an ionic strength of 0.5 M, a corresponding decrease of 0.10 pH unit (APKa = -0.10) could still be detected. The calculation ofthe electrostatic potentials in this subtilisin by the finite differ- ence Poisson-Boltzmann method (17) resulted in excellent agreement between the experimentally determined and pre- dicted ApKa values for His' (23). In a second example, a dramatic decrease of 1.5 pH units occurs in the pKa of His57 in bovine pancreatic trypsin when Asp102, to which His57 is hydrogen bonded, is replaced by an asparagine (31). No major structural rearrangements result from this substitution (32).
In RNase A, His"9 is found in a conformation that brings the side chains of His"9 (NW2) and Asp'12 (Q81) within hydrogen bonding distance (2.74 A) (33-35), whereas in both RNase-(1-118)-(111-124) and the asparagine analog, His"19 occupies predominantly a second conformation that is achieved by rotation around the Ca-C0 bond (8, 36). In this conformation, the distance between these two residues is considerably greater (9.9 and 8.8 A, respectively) (8, 18).
Biochemistry: Cederholm et al.
8120 Biochemistry: Cederholm et al.
Regardless of its positioning, however, a sizeable decrease in the pK. value for His119 was expected, and, indeed, the results from the application of the Poisson-Boltzmann equa- tion confirmed this expectation. In addition, these calcula- tions have revealed that the pK. shift for His12 is also substantially muted. The discrepancies between the observed and predicted
pK5 values for His12 and His119 may be the result of a number of factors. First, we have used the coordinates for crystal structures in 3 M ammonium sulfate to model the titration behavior of a protein in solution in 0.3 M NaCl. Second, with regard to His 19, the asparagine substitution has resulted in the imidazole ring ofthis residue undergoing a 1800 flip so that the N81 of the ring now forms a strong hydrogen bond to a water molecule (18). A third factor may be the effect of changes in the arrangement of bound water molecules. A comparison of the structures of RNase-(1-118)*(111-124) and D121N reveals numerous differences in the location and structure of crystallographically bound water networks (18). Such rearrangements may have resulted in significant changes in local dielectric constant. For RNase-(1-118).(111- 124) and D121N, the initial ApKa calculations included crys- tallographically bound water molecules, which were assigned a conventional dielectric constant of 2 (37). In both cases, large differences between the experimental (Table 2, rows 1 and 2) and theoretical (Table 2, rows 3 and 4) ApKa values were observed. However, when the crystallographically bound water molecules of the parent complex and- of the asparagine derivative were eliminated, there was a reduction in these discrepancies (Table 2, rows 5 and 6). This obser- vation suggests that the dielectric constant of bound water molecules may be closer to that of bulk solvent. In the case of lysozyme, better results were also obtained after removal ofbound water molecules from the crystallographic structure (38). When the coordinates for D121N are used in the presence
of crystallographically bound water, the discrepancy be- tween the observed and the predicted ApK. values (Table 2, rows 1 and 4) is moderated compared with the values obtained by using the coordinate set for the parent complex (Table 2, rows 1 and 3). This moderation suggests that the structure of the protein as a whole is accommodating (or attempting to accommodate) to the change in charge distri- bution resulting from the asparagine substitution. Such an accommodation results, therefore, in a multitude of small, but significant changes in structure throughout the molecule (18). Three ancillary resonances are seen in the proton NMR
spectra of D121N over the pH* range of 4.0-5.1 that are not seen with RNase A or with the parent complex; they do appear in the spectrum of free RNase-(1-118X, however (Fig. 1, spectrum E). The reduced binding energy between the asparagine-containing peptide and RNase-(1-118) indicated by this observation is supported by kinetic measurements at pH 6.0: Kd = 33 ,uM (vs. 1 FxM for the parent complex) (M. L. Ram and M.S.D., unpublished data). It is not likely that the presence of significant amounts of free RNase-(1-118) and [Asp121]RNase-(111-124) in the pH range 4.0-5.1 has seri- ously perturbed the titration curves of the histidine residues in the asparagine analog. If the pKa of the controlling group is 4.0 (as indicated by the presence of essentially equal concentrations of the complex and its two components at this pH value), only 7% of the chains remain dissociated at pH 5.1. Moreover, at pH 5.1, only 6% of His119 (pKa = 6.3) and 17% of His12 (pKa = 5.8) will have been titrated. The increase in Km and the reduction in kcat that accom-
pany the replacement of Asp121 by asparagine in RNase result in an enzyme that exhibits 6% activity against cytidine 2',3'-(cyclic)phosphate at pH 6.0 under standard assay con- ditions (refs. 3 and 4; M. L. Ram and M.S.D., unpublished
data). The present study has eliminated models in which this inactivation is associated with a drastic decrease in the ground state pKa value of active site His12 or His119, a distinct possibility a priori. Further measurements in the presence of active-site ligands may reveal differences in pKa values that would help to clarify the basis for the inactivation.
We thank Dr. Brian F. Pi Edwards, Dr. Philip D. Martin, and Dr. V. Srini J. deMel for providing the coordinates ofD121N. Amino acid analyses were performed by the Wayne State University Macromo- lecular Core Facility (supported in part by the Wayne State Univer- sity Center for Molecular Biology). This work was supported in part by the National Science and Engineering Research Council of Canada, the J. P. Bickell Foundation, The University of Windsor Research Board, and National Institutes of Health GrantGM 40630.
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