Histidine pKa shifts accompanying the inactivating Asp121 Asn
5
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 of Km 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 of RNase 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.
Histidine pKa shifts accompanying the inactivating Asp121 Asn
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.
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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