Proc. Natd. Acad. Sci. USAVol. 88, pp. 8116-8120, September 1991Biochemistry

Histidine pKa shifts accompanying the inactivating Asp121 Asnsubstitution 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 ofWindsor, 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 residues1-118 of RNase (obtained from the proteolytic digestion ofRNase A), and a synthetic 14-residue peptide containing resi-dues 111-124 of RNase, exhibits 98% of the enzymatic activityof 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. to2.7% of the value for RNase A. In the present work, lH NMRspectroscopy has been used to probe the ionization sates ofPis12, His 9, and His"' in this catalytically defective semisyn-thetic RNase. A comparison of the observed resonances ofD121N with those previously determined by others for RNaseA enabled us to assign the C2 proton NMR resonances toindividual residues; the asignment of Hisll9 was confirmed bytitrating 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 byusing a finite difference algorithm to solve the Poisson-Bodtzmann equation (the DELPH program, version 3.0) and arefined 2.0-A coordinate set for the crystal structure of D121Ndiffer 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 calulationsreduced, but did not reconcile, these discrepancies (His2,ApK. = -0.36; His"9, ApK. = -0.41).

combined with the corresponding peptide in which no aminoacid 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 residues111-118, is required to achieve both good binding and precisealignment of the two chains (7). A refined crystal structure at1.8-A resolution of RNase-(1-118)-(111-124), the fully activeparent complex, has been determined (8).The assignments of the C2 proton NMR resonances for

each of the four histidines in bovine pancreatic RNase A andtheir pKa values have been made in several laboratories(9-13). For a review, see ref. 14. The C2 proton NMRspectrum of semisynthetic RNase-(1-118).(111-124) and itspH dependence also have been obtained (15). The titrationbehavior of the four histidine residues in this semisyntheticderivative was indistinguishable from that found by others forRNase A. We report here the pH titration behavior of thehistidine residues in D121N.Using the solution to the Poisson-Boltzmann equation

provided in the electrostatics program DELPHI (16, 17) andthe coordinate sets for the crystal structures of both RNase-(1-118) (111-124) (8) and the asparagine analog (18), we havefound substantial differences between our experimentallydetermined 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 isinvariant throughout 40 species of mammalian pancreaticRNase (1), functions as part of the active site of bovinepancreatic RNase A (EC 3.1.27.5). Neutron diffraction anal-ysis of single crystals of RNase A has revealed the existenceof a hydrogen bond between the carboxyl Q81 of Asp12' andring N,2 of His119, a critical active site residue (2). Thereplacement of Asp'21 by asparagine in a semisyntheticderivative of RNase reduces kcat for the small substratecytidine 2',3'-(cyclic)phosphate at pH 6.0 to 12% of the valuefor RNase A and increases the value ofKm 4-fold (refs. 3 and4; M. L. Ram and M.S.D., unpublished data). To delineatefurther the role of Asp'21 in the function of RNase, we nowhave determined the apparent pKa values of three of the fourhistidine residues in the molecule by the measurement of thepH dependence of the C2 proton NMR resonances of thesemisynthetic derivative containing the Asn12' replacement.This derivative, "D121N," is prepared by combining RNase-(1-118), a totally inactive entity obtained by successivelydigesting 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 beenreplaced by asparagine (3, 4). If, instead, RNase-(1-118) is

MATERIALS AND METHODSMaterials. RNase A (RAF grade, salt-free, lot 54P6915)

used in the NMR experiments was purchased from CooperBiomedical. RNase A (type XII-A, lot 13F-8100) used in thepreparation of RNase-(1-118) was purchased from Sigma, aswere carboxypeptidase A (type I-DFP, lot 13F-8100) andpepsin (P-6887, lot 57F-8105, 4000 units/mg). 2H20,2HCH,NaO2H, and sodium 2,2-dimethyl-2-silapentane-5-sulfonatewere purchased from Merck Sharp & Dohme.

Preparation of RNase-(1-118). RNase-(1-118) was pre-pared by the successive digestion ofRNase A with pepsin andcarboxypeptidase 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-,umparticles; 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 residues1-118 of RNase A; RNase-(111-124), tetradecapeptide consisting ofresidues 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) andRNase-(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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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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by solid-phase synthetic methods (19, 20) and purified bymethods 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 derivedfrom aliquot samples of these stock solutions was dissolvedin 2H20, adjusted to pH* 3.0 with 1 M 2HC1, and then heatedto 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 insodium 2,2-dimethyl-2-silapentane-5-sulfonate and adjustedto the desired pH*. All pH* measurements given are thoseobserved directly and are not corrected for the deuteriumisotope effect. pH* measurements were made at room tem-perature with an Ingold 6030-02 microelectrode fitted to aCorning 240 pH meter. The proton NMR spectra wereacquired on a Bruker AC-300 NMR spectrometer at 30°Cwith a spectral width of 4500 Hz, 16,384 data points, andquadrature phase detection. The 1H2HO resonance was re-duced by homonuclear decoupling. The chemical shifts arereported with respect to the principal resonance of sodium2,2-dimethyl-2-silapentane-5-sulfonate.

Deuteration of [Asp'21JRNase_(111-124). The C2 proton ofHis"9 in the synthetic peptide [Asp'21]RNase-(111-124) wasfully deuterated by incubating a 16.8 mM solution of thepeptide 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 afour-parameter nonlinear least-squares curve-fitting programbased 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 theapparent acid dissociation constant, and n is the Hill coeffi-cient.

Calculation of Electrostatic Potentials. The predicted ApKavalues ofthe histidines in the semisynthetic RNase due to theD121N substitution were calculated by the application of afinite difference solution to a combination of the linearizedand nonlinearized Poisson-Boltzmann equations using theprogram DELPHI, version 3.0, on a Silicon Graphics 4D/70GTcomputer (23, 24).When the refined coordinates ofD121N (2.0-A resolution,

R = 0.187) (18) (Protein Data Bank, reference 2SRN) wereused, the N82 of Asn'2' was replaced by an O', and a chargeof - /2 was introduced at both 0o1 and O' of this newlyintroduced aspartic acid; the electrostatic potentials of eachof the histidines were then calculated based upon the loss ofthese two - Y2 charges at this residue. The sulfate anion,which is in the active site in the crystal structures, was notincluded in the calculations, but all crystallographicallybound water molecules were included. The following param-eters were used: ionic strength 0.3 M; protein interior andbound waters, dielectric constant of 2; solvent, dielectricconstant of 78.6; grid size, 60 x 60 x 60; focusing boundaryconditions 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 -1/2charges, assumed to be on O81 and O02 of Asp'1 , on the pKavalues for the histidines in the protein was calculated. Thesulfate anion was again eliminated, and all crystallographi-cally bound water molecules were included. The calculationsincluded 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. Forboth coordinate sets, the protein solvent boundary wasdefined by measuring the solvent-accessible surface (25, 26),

using a water probe radius of 1.8 A. One calculation using aprobe radius of 1.4 A reduced the ApKa values further by0.01-0.03 pH unit (see Table 2).

In separate computations, the crystallographically boundwaters of the parent complex and the asparagine analog wereeliminated and their corresponding ApKa values were calcu-lated.

RESULTS

Histidine 'H NMR Resonances of D121N. Spectrum A ofFig. 1 illustrates the 300-MHz proton NMR resonances ofthefour histidines of native RNase A in 0.3 M NaCl, pH* 4.0, at300C. This spectrum is in excellent agreement with previouslypublished data obtained under identical conditions (10).These four resonances have previously been assigned toHis'2, His"19, His'05, and His' in the order of decreasingchemical shift at pH* 4.0 (9-13). The analogous spectrum forthe parent semisynthetic complex, RNase-(1-118)-(111-124),spectrum B in Fig. 1, reveals a direct correspondence withthe 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 tripeptideGly-His-Gly and in RNase-(111-124) (spectrum C) (15). Thisresonance has been attributed to "unstructured" histidine,which is in slow exchange with those histidines in a nativeconformation. Spectrum E in Fig. 1 contains the NMRspectrum ofRNase-(1-118); the resonances at 8.88, 8.73, and8.34 ppm are due to His'2, His105, and His' by analogy withprevious studies (15). The additional resonance observed at8.6 ppm has again been ascribed to "unstructured" histidine;the reason for the broadness of this resonance, with twodistinct chemical shifts evident, is not clear.The corresponding spectrum for the asparagine analog,

D121N, in spectrum D in Fig. 1, contains two resonanceswith chemical shifts previously attributed to His'2 and His48in both RNase A and RNase-(1-118)-(111-124); these reso-nances, therefore, have been tentatively so assigned in thisanalog as well.

E

D

IB

19 105 48

A

9.0 8.8 8.6 8.4 8.2 8.08, 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 sampleswere at pH* 4.0. Spectra B, C, and D are fully relaxed; spectraA andE are partially relaxed. Shadings are explained in the text. Numbersin spectrum A are histidine positions.

.C

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The resonance due to His"9 was assigned by titratingD121N with a fully deuterated preparation of the tetrade-capeptide [Asp121]RNase-(111-124) at pH* 7.0. In a separateexperiment (data not shown), the addition of 1.5 equivalentsof the fully deuterated peptide to 2.8 mM D121N at pH* 7.08caused the resonance at 7.94 ppm, previously attributed toHis119, to decrease in intensity whereas those resonancesassigned to His12 (7.82 ppm) and His"05 (8.04 ppm) wereunchanged, confirming that the resonance at 7.94 ppm is dueto His"9.Three ancillary resonances (cross-hatched) appear in the

spectrum of D121N (spectrum D of Fig. 1) that are notobserved in the spectrum of RNase A (spectrum A) or in theparent complex (spectrum B), but which do appear in thespectrum of RNase-(1-118) alone (spectrum E). Their pres-ence in the spectrum of D121N suggests that the strength ofbinding between RNase-(1-118) and RNase-(111-124) may be

A

a

- ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~I

HU0

G0

U

-"%...F

J

reduced when asparagine replaces Asp'21; kinetic evidencesupports this postulate (see Discussion). These three reso-nances became progressively smaller and disappeared as thepH* was raised from 4.0 to 5.1, so their presence did notinterfere significantly with the tracing of the C2 resonancesduring the pH titrations (see below). Undeuterated[Asp'21]RNase-(111-124) (data not shown) exhibited thesame chemical shift as was seen in "unstructured" histidineat 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 Aand D121N in Fig. 2. The resonance of His"9 in the aspar-agine analog has been assigned as discussed above, whileHis'2 and His'05 have been assigned by comparison with thepreviously published proton NMR spectra of RNase A. Fig.3 A andB shows plots of the chemical shifts ofthe C2 protonsof His12, His105, and His"19 of RNase A and of D121N,respectively, as a function ofpH. Analysis of the pH titrationbehavior of His' was not possible due to the broadening ofthis resonance over the pH range and under the conditionsused in these experiments (27), a phenomenon that has beenobserved 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, arepresented in Table 1. In comparison with the pKa values ofRNase A, the pKa values of His12 and His 05 of D121Ndecrease by 0.18 and 0.16 pH unit, respectively, whenasparagine replaces aspartic acid at position 121. In contrast,the corresponding pKa value of His"9 is not altered by thissubstitution.Modeing the Electrostatic Effect of the ASp'21 -+ Asn

Substitution on the Histidine pK. Values. A refined coordinateset for D121N (18) was used in conjunction with a finite

9.08:8 8'6 8.4 8:2 8.0 i.8 768, ppm

EA.

A_ J D

C C

B

A~~~~~~~

8.8 8.6 84 8.2 8.0 7.8 7.68, ppm

FIG. 2. Spectra A-E, 300-MHz proton NMR spectra of 2.8 mMRNase at selected pH* values: A, 4.00; B, 5.04; C, 6.00; D, 7.01; andE, 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 0.3M NaCl at 30°C in 2H20.

8.5E

ce 8.0

7.53.5

9.0

8.5Ea

CO 8.0

4.5 5.5 6.5 7.5 8.5pH

7.5 . .

3.5 4.5 5.5 6.5 7.5 8.5pH

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 linesrepresent the calculated chemical shifts determined by the nonlinearleast-squares analysis described in Materials and Methods.

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Proc. Natl. Acad. Sci. USA 88 (1991) 8119

Table 1. Least-squares analysis of histidine titration profilesResidue* System PA6 pKa nHis'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 shownin 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 inthe pKa values ofthe three histidine residues as a result oftheasparagine substitution. The observed ApKa value of -0.16for His'05 is in good agreement with the predicted value of-0.10 for this residue (Table 2, rows 1 and 4). In contrast, thepredicted 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 and4).

If the numerous small structural changes with respect toprotein and crystallographically bound water that accompanythe substitution of asparagine for Asp12' (18) are ignored bycalculating electrostatic potentials using the coordinate setfor RNase-(1-118) (111-124) (8), the discrepancy between theexperimental and the predicted ApKa values for His12 andHis"9 resulting from the loss of two -1/2 charges at o01 andO02 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 betweenthe experimental and theoretical values, nevertheless, re-main.

Table 2. Comparison of the observed and predicted histidinePKa changes in the semisynthetic RNases

ApKa, pH units

Row His'2 His'05 His"19 Comments1* -0.18 -0.16 -0.022t -0.09 -0.12 +0.053* -0.85 -0.15 -1.1 Asp'2'§, +H2014O -0.58 -0.10 -0.55 Asn'21i, +H20¶51t -0.39 -0.09 -0.41 Asp'21§, -H2016t -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.

DISCUSSIONThe similarity of the chemical shifts of fully protonated andfully 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 residuesare similar in all three molecules at very low pH and at veryhigh pH. Even at pH* 4.0, the proton NMR spectrum ofD121N contains resonances that correspond well with thoseofHis'2 and His' in the parent complex and in RNase A (Fig.1). At this pH* value, however, the chemical shifts of His'05and His"19 in the asparagine analog are significantly different.The substitution of asparagine at position 121 has alsoresulted in a decrease in the observed pKa values ofHis12 andHis'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 thismutation.Some decrease in the pKa values of the histidine residues

was anticipated, as the replacement of aspartic acid byasparagine removes a negative charge from the molecule; thischange would be expected to destabilize the positivelycharged protonated form of a histidine residue and concom-itantly decrease its pKa value. Such an electrostatic effect issharply dependent upon distance and ionic strength, but ithas been shown experimentally to remain detectable atconsiderable distances and substantial ionic strengths. In anextracellular 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 siteHis' 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 ionicstrength of 0.5 M, a corresponding decrease of 0.10 pH unit(APKa = -0.10) could still be detected. The calculation oftheelectrostatic potentials in this subtilisin by the finite differ-ence Poisson-Boltzmann method (17) resulted in excellentagreement between the experimentally determined and pre-dicted ApKa values for His' (23). In a second example, adramatic decrease of 1.5 pH units occurs in the pKa of His57in bovine pancreatic trypsin when Asp102, to which His57 ishydrogen bonded, is replaced by an asparagine (31). Nomajor structural rearrangements result from this substitution(32).

In RNase A, His"9 is found in a conformation that bringsthe side chains of His"9 (NW2) and Asp'12 (Q81) withinhydrogen bonding distance (2.74 A) (33-35), whereas in bothRNase-(1-118)-(111-124) and the asparagine analog, His"19occupies predominantly a second conformation that isachieved by rotation around the Ca-C0 bond (8, 36). In thisconformation, the distance between these two residues isconsiderably greater (9.9 and 8.8 A, respectively) (8, 18).

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Regardless of its positioning, however, a sizeable decrease inthe pK. value for His119 was expected, and, indeed, theresults 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 alsosubstantially muted.The discrepancies between the observed and predicted

pK5 values for His12 and His119 may be the result of a numberof factors. First, we have used the coordinates for crystalstructures in 3 M ammonium sulfate to model the titrationbehavior of a protein in solution in 0.3 M NaCl. Second, withregard to His 19, the asparagine substitution has resulted inthe imidazole ring ofthis residue undergoing a 1800 flip so thatthe N81 of the ring now forms a strong hydrogen bond to awater molecule (18). A third factor may be the effect ofchanges in the arrangement of bound water molecules. Acomparison of the structures of RNase-(1-118)*(111-124) andD121N reveals numerous differences in the location andstructure of crystallographically bound water networks (18).Such rearrangements may have resulted in significantchanges in local dielectric constant. For RNase-(1-118).(111-124) and D121N, the initial ApKa calculations included crys-tallographically bound water molecules, which were assigneda conventional dielectric constant of 2 (37). In both cases,large differences between the experimental (Table 2, rows 1and 2) and theoretical (Table 2, rows 3 and 4) ApKa valueswere observed. However, when the crystallographicallybound water molecules of the parent complex and- of theasparagine derivative were eliminated, there was a reductionin these discrepancies (Table 2, rows 5 and 6). This obser-vation suggests that the dielectric constant of bound watermolecules may be closer to that of bulk solvent. In the caseof lysozyme, better results were also obtained after removalofbound 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 valuesobtained by using the coordinate set for the parent complex(Table 2, rows 1 and 3). This moderation suggests that thestructure of the protein as a whole is accommodating (orattempting to accommodate) to the change in charge distri-bution resulting from the asparagine substitution. Such anaccommodation 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 notseen with RNase A or with the parent complex; they doappear in the spectrum of free RNase-(1-118X, however (Fig.1, spectrum E). The reduced binding energy between theasparagine-containing peptide and RNase-(1-118) indicatedby this observation is supported by kinetic measurements atpH 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 thepresence 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 residuesin the asparagine analog. If the pKa of the controlling groupis 4.0 (as indicated by the presence of essentially equalconcentrations of the complex and its two components at thispH value), only 7% of the chains remain dissociated at pH5.1. Moreover, at pH 5.1, only 6% of His119 (pKa = 6.3) and17% 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 resultin an enzyme that exhibits 6% activity against cytidine2',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 thisinactivation is associated with a drastic decrease in theground state pKa value of active site His12 or His119, a distinctpossibility a priori. Further measurements in the presence ofactive-site ligands may reveal differences in pKa values thatwould 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 acidanalyses 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 partby the National Science and Engineering Research Council ofCanada, the J. P. Bickell Foundation, The University of WindsorResearch Board, and National Institutes of Health GrantGM 40630.

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