Int. J. Peptidehotein Res. 14, 1979, 199-212

A NEW ARYLATING AGENT, 2-CARBOXY-4,6-DINITROCHLOROBENZENE

Reaction with Model Compounds and Bovine Pancreatic Ribonuclease

JAKE BELLO, HERBERT IIJIMA and GOPINATH KARTHA

Department o f Biophysics, Roswell Park Memorial Institute, Buffalo, New York, USA

Received 11 October 1978, accepted for publication 7 March 1979

The reagent 2-carboxy-4,6.dinitrochlorobenzene (CDNCB) reacts with the imino, amino and sulfhydryl groups o f model compounds. A t p H 8.2, sulfhydryl groups react much faster than do amines. Na-Acetylhistidine, N"-acetyltyrosine and Na- acetyltryptophan d o not react. Poly (L-Lysine) and poly (or,-Iysine) react about 50 times as fast as does Na-acetyllysine. A dichloroanalog. 6-carboxy-2,4-dinitro- 1,3-dichlorobenzene, shows stepwise reactivity with amines. With bovine pancre- atic ribonuclease, which contains no sulfhydryl, CDNCB reacts preferentially with the €amino o f Lys-41 a t 450 times the rate with the €-amino o f Na-acetyl- lysine. The preferential reactivity at Lys-41 is discussed in relation to the p K of Lys-41, the cationic character of the active site cleft, and the mechanism of RNAase action on substrates.

Key words: active site; 2carboxy4,6dinitrochlorobenzene; lysyl-41; ribonuclease.

Arylating agents are of value in exploring protein fine structure. Hirs et al. (1965) and Hirs & Kycia (1965) used FDNB, and Carty & Hirs (19680, b) SNFB, for studying bovine pancreatic ribonuclease. Goldfarb et al. (1 974) studied the reactivity of the lysyl residues of RNAase toward TNBS. To increase the selec- tivity of arylation, we considered the use of a

Abbreviations: FDNB, l-fluoro-2,4dinitrobenzene; SNPB, 4-sulfonyloxy-2-nitrofluorobenzene; CDNCB, 2tarboxy4,6dinitrochlorobenzene; TNBS, 2,4,6-tri- nitrobenzene sulfonic acid; RCM-, reduced-carboxy- methylated; DNP-, 2,4dinitrophenyl; SNP-, 4-sulfonyl- oxy-2-nitrophenyl; CDNP-, 2-carboxy4,6,-dinitrophe- nyl; TNP-, 2,4,6-trinitrophenyl; RNAase I, bovine pancreatic ribonuclease (EN 1972, 3.1.4.22), formerly called RNAase A (Ec 2.7.7.16); CM, carboxymethyl (on histidyl residue); RCM, carboxymethyl on sulf- hydryl formed by reduction of disulfide.

reagent of intrinsically low reactivity toward amines which are under no activating influence. We found 2carboxy4,6-dinitrochIorobenzene (CDNCB) to be relatively sluggish toward small model compounds, but to have high reactivity and high selectivity toward a specific lysine residue in RNAase. It is commercially available and easy to purify, is water soluble and slow to undergo hydrolysis. The carboxyl group can be converted readily to uncharged derivatives, such as esters and amides, containing different chain lengths, reporter groups, etc. Some reactions of CDNCB with RNAase modified on arginine have been reported earlier (Iijima et d, 1977). There is commercially available the isomer 4- carboxy-2,6-dinitrochlorobenzene; while this manuscript was in preparation Brautigan et al. (1978) published a study of cytochrome C with this isomer. A commercially available dichloro-

0367-8377/79/080199-14 $02.00/0 0 1979 Munksgaard, Copenhagen 199

J . BELL0 ET AL.

analog, 6carboxy-2,4-dinitro-l,3-dichloroben- zene, is a potential cross-linker,

MATERIALS

Phosphate-free RNAase, type RAF, and yeast RNA were from Worthington Biochemical Corp., Lys-Lys-Lys from Cyclo Chemical Co., poly(L-lysine .HBr) from Miles Laboratories, poly(DL-lysine .HBr) from Sigma Chemicals, and NQ-acetyl-L-lysine from Fox Chemical Co. The CDNCB (sold as 2-chloro-3,5dinitrobenzoic acid) from Aldrich Chemical Co. was recrystal- lized twice from acetonecyclohexane; the nearly-white cyrstals melted at 193-194" (un- corrected). 6-Carboxy-2,4-dinitro-l,3-dichloro- benzene was purchased from Aldrich Chemical Co. TPCK-trypsin, "essentially freed of chymo- trypsin", was purchased from Calbiochem. h i n e x 50W-X2 resin is a product of Bio-Rad Laboratories. His-1 19-carboxymethyl-RNAase was prepared according to Fruchter & Crestfield (1967).

METHODS

Rates of reaction of CDNCB with model compounds A solution 1 mM in both CDNCB and model compound was maintained at pH 8.2 (pH-stat), and the absorbance at 370nm was read at 2-10-min intervals.

N '-CDNCB-P-acetyllysine Na-Acetyllysine (120 mg, 0.9 mmol), 143 mg (0.58mmol) CDNCB, and 172 mg (2.6mmol) NaHCO, were dissolved in 1.5 ml H2 0; after the foam subsided the reaction vial was capped. After 8 days the reaction mixture was passed through a 1.5 x 60cm column of Bio-Gel P-2, with elution by water. The yellow material appearing between 75 and 145ml was pooled and lyophilized, dissolved in lOm1 of water, and acidified with 1 N HC1. The product was extracted twice with 15-ml portions of ether. The ether was evaporated and the yellow residue was converted to the sodium salt with the minimum amowt of 0.1 N NaOH, and lyophilized. Purit was not determined, but

from the method of synthesis.

200

eX9 was 14 x 10 r and the identity is inferred

CDNP-methylamine To 0.33 g (5 mmol) methylamine hydrochloride in 25ml water were added 1.5g NaHCO, and 1.25 g (5 mmol) of CDNCB. The reaction was run at 40" for 4h, then at room temperature overnight. Acidification to pH 2 with S N HCl yielded 0.70g of yellow precipitate (75% of theory), which was recrystallized four times from 75% ethanol. Analysis for C, H and N agreed with theory. The absorption spectrum of the product in 0.1 M sodium phosphate (pH 7) gave €370 = 15 x 1O3M-'cm-'.

CDNP-gy cine To 0.75 g (10mmol) glycine and 2.4g NaHC0, in 25 ml water was added 2.22g (9 mmol) of CDNCB in 10 ml ethanol. During stirring for 2 days at room temperature the yellow sodium salt of the product precipitated. The precipitate was dissolved by warming the reaction mixture. The warm solution was acidified to pH 1.5-2 with S N HCl. On cooling, yellow crystals separated. Yield, 2.3 g (80%). After recrystal- lization four times from 95% ethanol and once from 20% ethanol, the m.p. was 213-214'. Analysis for C, H and N agreed with theory. The spectrum in 0.1 M sodium phosphate (PH 7) gave €370 = 15 x lo3 M-'cm-'.

Reaction of CDNCB with p o l y f ~ - l y s i n e ) To 109mg (0.52mmol lysine residue) poly(L- lysine * HBr) in 50 ml H 2 0 was added 42 mg (0.17mmol) CDNCB. The pH was adjusted to 8.0 with 1 N NaOH. After 3 h, when about 72% of the CDNCB had reacted (taking €370 of the product as 15 x lo3), equivalent to 23% of the lysyl residues, the reaction mixture was passed through a 2 x 80cm column of Sephadex (3-25 and eluted with 0.5 M acetic acid.

CDNP-R NAase To S6mg RNAase in 1.75ml H 2 0 was added 0.25ml CDNCB solution (14.4mM, in 0.015 N NaOH). The mixture was maintained at pH 8.2 on a pH-stat at room temperature for 2 h. Ary- lation was terminated by adjustment of the pH to 6.0. The mixture was then immediately applied on a column of Bio-Rex 70 (Fig. 1). The major yellow product (CDNP-RNAase) was eluted at 80-11Oml. The product was then desalted on a 2.54 x 60cm column of Sephadex

ARY LATION OF RIBONUCLEASE

15

I C Y c) z a m

m SI oe

EFFLUENT rnl

FIGURE 1 Separation of CDNP-RNAase on a 0.9 X 110-cm column of Bio-Rex 70 (100-200 mesh), with 0.2M sodium phosphate (pH 6.1, 30°, 10 ml/h). Absorbance at 280nm. 0; 370nm, 0. The product peak is at 100 ml; reagent and unmodified RNAase emerged at 45 and 330 ml, respectively.

G-25, with 0 . 0 2 ~ ammonium acetate, and lyophilized. 25 mg CDNP-RNAase was recovered.

Reduction and carboxymethylation o f disulfide The disulfides of RNAase and CDNP-RNAase were reduced with dithiothreitol in 8 M urea, and the sulfhydryls were carboxymethylated with bromoacetic acid (Bello & Nowoswiat, 1969) at pH 8.3.

Tryptic digestion and peptide analysis The RCM-RNAase and RCM-CDNP-RNAase were digested with trypsin in 0.2M sodium phosphate buffer at pH 7.0 (Hirs et al., 1956). The digests were analyzed on a Technicon peptide analyzer using a 0.6 x 95cm column of Aminex 50W-X2 for chromatographic separ- ations. The column was operated at 40", and elution was carried out with the buffer gradient system (Instruction Manual T-67-101, p. 27, Technicon Corp., Ardsley, NY 10502). The analyzer manifold was modified to deliver 60% of the column effluent to a fraction collector; tubes 2, 3 and 5 were changed to tubes of I.D. 0.015,, 0.020 and 0.045 inch, respectively. Fractions of 3ml were collected. Ninhydrin color produced in the base-hydrolyzed line is

shown in the Figures. Effluent from experimen- tal samples was also read at 370 nm.

Amino acid analysis Samples were hydrolyzed under nitrogen at 1 10" for 24 h in sealed tubes, then evaporated at 35" in a stream of nitrogen.

X-Ray diffraction CDNP-RNAase was crystallized by our usual procedure (King et al., 1956) and analyzed by X-ray diffraction. The crystals are monoclinic with space group and have the cell para- meters: a = 30.16A, b = 38.72 A, c = 53.17 A and B = 105.60". These values are close enough to the parameters (a = 30.13 A, b = 38.1 1 A, c = 53.29 A and 0 = 105.75") of the unmodi- fied protein for the possibility of examining the site of modification by the electron density difference method.

Three-dimensional diffraction data of CDNP- RNAase were collected with fdtered CuKa radiation to a Biagg angle of 15" corresponding to a resolution higher than 3 A, by the stationary crystal, stationary counter method, and an approximate absorption correction was applied in addition to the usual geometrical corrections to evaluate the structure amplitudes, IFpHI, of the reflections from the modified crystals. These amplitudes were scaled against the native protein structure amplitudes, IFpl, to take account of the differences in crystal sizes as well as overall thermal parameters causing differing rates of intensity decay at increasing scattering angles. Comparison of the two sets of structure amplitudes, IFpHl and IFpl, gave an overall agreement residual R = 0.07. This value of 7% in R is the same order that is obtained for two sets of data from the same type of crystals and is thus much smaller than the 15-30% one obtains for comparison between the free protein and heavy atom derivative crystals used in structure determination. Knowledge of the detailed electron density distribution in the free protein crystals permits us to search for specific electron density differences between the protein and modified isomorphous derivative crystal. The phase angles and structure amplitudes of the diffracted X-ray beams for the native protein, together with the structure amplitudes of the modified protein, were used in computing a

20 1

J. B E L L 0 ET AL.

point-by-point difference electron density map between the two molecules throughout the unit cell of the crystal using the equation:

(XYZ) = { IFPHI - IFPI}

exp - {2ni(hx + ky + lz) - (1lhk1)

The difference densities were computed at intervals smaller than 0.5 A along the three axes and contoured in sections parallel to the a-b plane. The map was mostly featureless, con- firming the initial assumption of close isomor- phism between the two crystals. A projection of the difference density of a small region of the map showing the largest change is shown in Fig. 5. To facilitate the location of the region of the largest density change, which presumably corresponds to the site of attachment of the CDNPgroup, a similar projection of the electron density of the native molecule in this region is given. Some of the residues in the native mol- ecule are unsuitable for estimation of relative peak heights. This is due to the fact that while the electron density map of the native protein is based on diffraction data to 2 A resolution and protein phases evaluated using seven derivatives, the map for CDNP-RNAase was computed with 3 A data, and in addition assumed that the phases of the native and CDNP ribonuclease crystals are basically the same. On both these counts, the density of the CDNP group in the difference map is much lower than in the native map. For clarity the contour levels of the difference map were plotted at about l/lOth the level of the contours in the native protein map and are thus much lower than would appear by direct inspection.

RESULTS

Reactions with amino acids and other model compounds; spectrum, stability to hydrolysis and light Crystalline derivatives of methylamine and glycine were obtained, which gave the theoreti- cal C, H and N contents. Table 1 shows that the amino and imino groups of the common amino acids react with CDNCB at pH9. The rate of hydrolysis of CDNCB is negligible at pH9. There is a considerable degree of regeneration of amino acids with 6 N HCl at 106", the amount varying with the amino acid. Table 2

202

TABLE 1 Reaction of CDNCB with amino acid mixture"

Amino Before hydrolysis % Regeneration acid % modified on hydrolysis

ASP Thr Ser Glu PI 0

Gly Ala Val f cys Met Ile Leu Tyr Phe LY s His k g

56 70 98 76

100 100 99 65 93 71 71 66 81 75 94 93 99

18 64 21 30 19 31 20 85 66 78 69 5 2 73 67 71 50 51

* To 1 ml of the Bio-Rad calibration mixture (2.5 pmol of each amino acid, total 43pmol of amino group) were added 2.6 ml water and 9.1 mg (37pmol)CDNCB. The pH was adjusted to 9.0 with 1 N NaOH and the volume brought to 4.5 ml. The pH was adjusted occasionally during the 14day period of reaction. Aliquots of the reaction mixture, before and after acid hydrolysis, were subjected to amino acid analysis. No corrections were made for losses of serine and threnonine.

shows that lysine reacts 15 times as fast as does N"-acetyllysine. Table 1 shows that 71% of lysine is regenerated by acid. Hydrolysis of N E - CDNP-NQ -acetyllysine resulted in only 15% regeneration of lysine. Thus it appears that in lysine the a-amino is more reactive toward CDNCB, but that the e-amino derivative is more stable than the a-amino derivative toward cleavage of the N-phenyl bond. The CDNP- derivatives have spectra typical of nitroanilines, with maxima at 368-370nm in phosphate buffer (PH 7) and at 345-350 nm in 0.1 M HC1. Exposure of an aqueous solution in Pyrex to 30 h of fluorescent room light caused no change in the absorption spectrum of N'-CDNP-N"- acetyllysine.

Reaction rates Analysis of the data for formation of CDNP- derivatives of several small model compounds

ARYLATION OF RIBONUCLEASE

TABLE 2 Rate constants of the reactions of CDNCB with

model compounds and ribonucleasp

the protonated amino group. In addition, inter- actions between the carboxylate of CDNCB and the ammoniuni group would be expected to favor the reaction with mercaptoethylamine. With cysteine, ninhydrin analysis on aliquots of the reaction mixture showed that 40%- of the

Substance lo4 X k(M-' sec-l )b

Ribonuclease Mercaptoethylamine Cysteine Mercaptoethanol Dithiothreitol ~o ly -~ -~ys ineC*d Poly-DL-lysineCVe Lysine Cystine Lys-Lys-Lys Methylamine Arginine Glycine Gly-GlyGlyGly amide Na-AcetyllysineC Proline N"-Acetylhistidine NO-Acet yltr yptophan Na-Acet yltyrosine

900 1333 560 25 3 160 100 90 31f 15' 9g 9 4 3 2 2 1 0 0 0

a Except as noted, the concentration of CDNCB and model compound was 1 mM in each case, a pH of 8.2 was maintained on a Radiometer pH-stat, and the absorbance at 370nm was read at 2-10-min intervals in a Bausch & Lomb Spectronic 20. Reactions at 27". Using a molar extinction coefficient of 15 X

10' M - ~ cm-l. Reaction carried out under conditions different from

those of the other substances in this table. The pH was maintained manually at 8.0-8.2, except for N%cetyl- lysine at about pH9.2. The rate constants for poly(DL4ysine) and poly(L-lysine) were calculated using the total concentration of lysyl residues.

Degree of polymerization about 120; hydrobromide. Degree of polymerization about 250; hydrobromide. Calculated per mole of amino acid. Calculated per mole of peptide; 2.2 X lo-'' M-' sec-l

per mole of amino group.

showed the reactions to be second order kinetically. Table 2 shows that mercaptoethy- lamine reacts faster than any other model compound tested. Ninhydrin analysis of the reaction mixture showed no decrease in ASm up to 1 h, indicating that only the sulfhydryl group reacted. Mercaptoethylamine reacted faster than did mercaptoethanol, probably because of a lower pK for the sulfhydryl of the former, resulting from the positive charge of

reaction occurred at the amino group. N"- Acetyl derivatives of tryptophan, histidine and tyrosine do not react with CDNCB at pH 8.2; but the possibility of reaction at activated residues in some proteins is not excluded.

The data for glycine and Gly-Gly-Gly-Gly amide showed no important difference in observed rate between a-amino groups of free amino acids and peptides. Adjusting for the difference in the fraction of unprotonated amino groups (pK values of 9.78 and 7.75 respectively, for a-amino of glycine and oligo- glycines, Edsall & Wyman, 1958) the intrinsic reactivity of glycine is 100 times that of Gly- Gly-Gly-Gly amide, indicating that the a-amino' of the peptide is a weaker nucleophile, presum- ably because of the inductive effect of the peptide group. The rates for poly(L-lysine) and pOly(D L-lysine) were calculated on the assump- tion of second order kinetics and on the total concentration of lysyl residues. Poly(L4ysine) and poly(D L-lysine) reacted faster than did the model amines of Table 2, 50 times as fast as N"-acetyllysine (probably faster if the pH values had been the same) and 10 times as fast as Lys-Lys-Lys. The rate of reaction of RNAase with CDNCB was proportional to the product of both concentrations. The second-order rate constant for RNAase was much larger than for nonsulfhydryl models, indicating the presence of a highly reactive amino group in this enzyme.

Reaction of CDNCB with RNAase carboxy- methylated on His-119 was much slower than with RNAase, although accurate kinetic data were not obtained. The anionic carboxymethyl group on His-1 19 presumably inhibits binding and/or orientation of CDNCB.

6-Carboxy-2,4-dinitro-I ,3dichlorobenzene A preliminary study of the reaction of this difunctional analog with glycine was done with a 1 : 1 and with a 20: 1 molar ratio of glycine to reagent. Products were not isolated, but at the 1 : 1 ratio the spectrum had the usual A,, = 370nm, and with excess glycine the spectrum had A,, = 410nm. A reaction with a large

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J . BELL0 ET AL.

excess of methylamine gave A,, = 420nm. The spectrum of the 1 : 1 reaction mixture with glycine showed no peak or shoulder at 41 0 nm, indicating that a mono-, and not a bis-glycine, derivative of CDNCB was formed. The results with low and high ratios indicate that both chlorine atoms can be replaced but one is more reactive than the other. The difference in reac- tivities between the two C-Cl bonds suggests the possibility of controlled stepwise reactions to produce a cross-link between different kinds of proteins, or to arylate a protein and to follow with a reaction at the second C-Cl to introduce another functional group or reporter group.

Preparation and analysis of CDNP-RNAase RNAase was treated with a slight deficiency of CDNCB (1.0:9 molar ratio) and the reaction was carried to 69% of completion (indicated by base uptake in the pH-stat). The chromatogram (Fig. 1) showed a single major yellow fraction, and a small yellow fraction (about 7% of total yellow product). The latter substance exhibited enzymic activity nearly equal to that of RNAase; thus the modified residue is not essen-

tial to catalysis. This minor product was not studied further; by analogy with the reaction of RNAase with SNFB (Carty & Hirs, 1968), FDNB (Hirs & Kycia, 1965) and TNBS (Goldfarb et al., 1974), this product may be modified on the a-amino of Lys-1 .

The spectrum of the major product in 0.1 M sodium phosphate (pH 7.5) shows maxima at 277 and 378 nm, with E values of 15 x lo3 M cm-' and 15.8 x 1O3M-'cm-', respectively (Fig. 2). The latter is 6% larger than that of the products from methylamine and glycine.

The enzymic activity of the product on yeast RNA, at pH 6 and 7.5 was only 0.6% of that of RNAase. This low enzymic activity indicates that the residue modified is in or near the cata- lytic site. This result points immediately to Lys- 7 or Lys-41, which are in the active site, according to the X-ray structure of RNAase- ligand crystals (Kartha et al., 1968).

The site of modification was located by tryptic digestion of reducedcarboxymethylated CDNP-RNAase (RCM-CDNP-RNAase). After 2 h of digestion, the chromatogram showed loss of only the 14h peak (shaded area in Fig. 3), present in the digest of RCM-RNAase, and the

I ' ' -1

\

x ! / \ I /

1 ---L__

350 4 00 450 500 -~ vase I

300 O L ' - .

250

WAVELENGTH, nrn FIGURE 2 Spectra of RNAase I and CDNP-RNAase in 0.1 M sodium phosphate, pH 7.5. Concentrations, corrected for 10% moisture in lyophilized proteins, were 0.9 mg/ml for CDNP-RNAase. Recorded on a Cary 15 spectrophotometer with the 1.0 slidewire.

204

, i I '. I I I I J

2 8 12 16 20 0

TIME, HOURS

FIGURE 3 Peptide analysis of a 2-h digest. 12 mg RCMCDNP-RNAase was digested with 0.06 mg TPCK-trypsin for 2 h at pH 7.0 and 27". Solid line: ninhydrin color at 570nm. Dashed line: absorbance at 370nm. Shaded area present in control sample but absent in experimental sample.

appearance of two yellow peaks. The 14-h peak from the control was lyophilized, and freed of salt and contaminating peptides on a column (0.9 x 60cm) of Sephadex G-15. Amino acid analysis (Table 3) showed it to be RCM-Tryp 9, residues 40-61. Since the yellow peaks were highly contaminated with other peptides, they were not analyzed. Instead a 20-h tryptic digest was chromatographed on the same resin (Fig. 4) and two yellow peptides were obtained. Passage of each colored material through the Sephadex G-15 column removed salt and contaminating peptides. Amino acid analyses (Table 3) showed the first yellow peptide to contain the sequence CM-Cys-40 through Phe46; the second, CM-Cys40 through Asn-44. Thus, only one lysine can bear a CDNP group, namely Lys-41, which is not a site of tryptic cleavage because of the adjacent prolyl residue.

Cleavage at phenylalanyl and asparaginyl indi- cates chymotryptic contamination of the trypsin preparation. Chymotrypsin has been known to cleave bonds at asparaginyl residues (Cha & Scheraga, 1963), but does not cleave at Asn44 of native RNAase. It is not known why the CDNP group at Lys-41 facilitates cleavage at Asn-44.

An arylation of RNAase was also done with six moles of CDNCB per mole of protein. The absorbance-time curve showed that only one CDNP-residue is incorporated at a significant rate, the reaction becoming very slow as the amount of CDNP incorporated approached one residue per molecule of RNAase. This indicates that all but one lysyl side chain are sluggishly reactive even when there is reagent present in addition to that which may be bound in the active site. The product chromatographed ident-

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ically with that obtained with a deficiency of CDNCB, with the same ratio of major product to minor product.

Effect of inhibitors The three inhibitors of RNAase activity listed in Table 4 showed that UMP, the most effective of the three as an inhibitor of enzymic activity, is also most effective inhibitor of the reaction between CDNCB and RNAase. The inhibitors do not react with CDNCB.

Binding of CDNP-derivatives to RNAase The activity of RNAase (on C > p at pH 7) was not affected by NE-CDNP-Na-acetyllysine when this derivative was present at up to 24 times the molar amount of enzyme.

A solution of poly(L1ysine) containing 7 pmol CDNP-lysyl residues (25% of its residues modified) and RNAase, 0.7pmo1, was placed on a 1.5 x 80cm column of Sephadex G-100, and eluted with 0 . 0 2 ~ ammonium acetate, pH6.1. About 90% of the RNAase was recovered in a colorless fraction at the same

elution volume as native RNAase. Thus, binding of CDNP-derivatives to RNAase must be weak.

X-Ray crystallography CDNP-RNAase was crystallized in the presence of phosphate. RNAase does not crystallize in the absence of an anionic active-site ligand. The only known exception is the crystallization of RNAase carboxymethylated on His-1 19 (Bello & Nowoswiat, 1969), where the carboxylate of the carboxymethyl appears to fulfil the require- ment. (This His-1 19-CM-RNAasecrystal is meta- stable, and undergoes a spontaneous transition to another form unless phosphate is present.) Data on CDNP-RNAase were collected to a resolution of 3 A, and these crystals were found to be substantially isomorphous with standard RNAase crystals. A difference electron density map between native and CDNP-RNAase was mostly featureless (Fig. 5). The largest density difference was adjacent to the site (in the native crystal) of the terminus of the side chain of Lys-41, and was within the active site cleft. In addition, near the region at which phosphate ion is normally situated in the crystal of native

TABLE 3 Amino acid analyses of modified ribonuclease and tryptic peptides

Amino RCMCDNP-RNAase 14-h peak, Fig. 3 9-h peak, Fig. 4 11-h peak, Fig. 4 acid Obtained Theorya Obtained Theoryb Obtained TheoryC Obtained Theory'

CM-Cys 8.3 8 0.9 2 0.9 1 0.9 1 ASP 14.6 15 2.3 2 1 .o 1 1 .o 1 ThI 10.0 10 1.3 1 1 .o 1 0.2 -

- - 0.1 - Ser 13.9 15 2.2 2 Glu 12.3 12 3.0 3 Pro 3.9 4 0.9 1 0.9 1 1 .o 1 G ~ Y Ala 11.7 12 2.2 2 Val 9.2 9 3.0 4 1 .o 1 0.9 1 Met 3.6 4 Ile 2.4 3 Leu 2.2 2 0.8 1

- - - 0.2 - Tyr 6.2 6 0.2 Phe 3.2 3 0.8 1 1 .o 1 0.2 - LYS 9.2 10 1.9 2 0.2 1 0.2 1 His 4.0 4 1 .o 1 Arg 4.2 4

- - - -

- - - - - 4.4 3 0.1 - - - -

- - - - - -

- - - - - -

- - - -

- - - - - - - - - -

________ ~______ ~ _ _ _ _ _

a For RCM-RNAase. For the peptide of residues CMCys-40 to Lys-61. For the peptide of residues CMCys-40 to Phe-46. For the peptide of residues CMCys-40 to Asn-44.

206

ARY LATION OF RIBONIJCLEASE

I .5

I .o W 0 z

[L

a m

m 2 a 0.5

EFFLUENT ml

FIGURE 4 Peptide analysis of a 2011 digest. 12 mg RCMCDNP-RNAase were digested with 0.06 mg TPCK-trypsin for 20 h at pH 7.0 and 27". Solid line: ninhydrin color at 570 nm. Dashed line: absorbance at 370 nm.

RNAase, there are some positive and negative densities indicative of a slight displacement of the phosphate in the crystal of CDNP-RNAase. All other changes in the electron density map were of smaller magnitude. The CDNF' group does not occupy the pyrimidine or ribose binding region, but is in the wider space leading to the binding region, in the same general region as the DNP group in DNP-RNAaseS (Mewell er ul., 1973). Binding interactions in this larger space are expected to be weaker, in accord with our data indicating weak binding and weak enzymic inhibition by CDNP derivatives.

DISCUSSION

The higher reaction rates for poly(L-lysine) and poly(DL-lysine) compared with those of small molecules are presumed to result from the higher density of cationic groups to bind the

carboxylate of CDNCB, and of unprotonated reactive amino groups, resulting in a cooperative reaction. Also, the lysyl residues first titrated have relatively low pK values (Ciferri er ul.,

TABLE 4 Effect of enzymic inhibitors on the reaction of

CDNCB with ribonucleasea

Inhibitor Rate, % of control

Uridine, 0.4 mM 86 Sodium phosphate, 0.4 mM 76 Sodium phosphate, 4 mM 29 UMP (2' & 3'), 0.4 mM 62 UMP (2' & 3'),4mM 15

a Concentrations of CDNCB and RNAase were 0.4 mM. Reaction mixtures were maintained on a Radiometer pH-stat set at pH 8.2, and production of yellow color was followed by measuring A,,,, at 2-lO-min inter- vals, with a Bausch & Lomb Spectronic 20.

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J . BELL0 ET AL.

1968). One might have expected that Lys-Lys- Lys would be more reactive than lysine, but the reverse was the case.

Comparison with other alkylating agents FDNB reacts preferentially with Lys-41 of RNAase, at 10 times the rate of the reaction at the a-amino of Lys-1 and 70 times the rate of reaction with the €-amino groups of model peptides (Hirs et al., 1965; Hirs & Kycia, 1965). SNFB was reported to produce about equal amounts of modification at Lys41 and at the a-amino of Lys-1 , although the pH-independent rate constants were in the ratio of 16: 1 (Carty & Hirs, 1968a,b). Thus, the uncharged FDNB and the anionic CDNCB show high preference

J b

for Lys-41 while the anionic SNFB appears not to show so strong a preference. The result for SNFB arose from the use of an excess of SNFB and from the pH of the reaction mixture. In our reaction of RNAase with a 6 : 1 ratio of reagent to RNAase, the relative amounts of reaction at Lys-41 and at the second site were the same as at 0.9:l reactant ratio. At a suf- ficiently high CDNCB ratio, at which reaction with Lys-41 would become pseudo-first order and the active site would become saturated, we would expect proportionately more of the second product.

Such a situation appears to exist in the results of two reports by Goldfarb on TNBS. In the first (Goldfarb, 1974), the rate of reaction of

S U L L IN i - 0 a I0

FIGURE 5 Left: Projection of difference electron density (contours in arbitrary units) between RNAase I and CDNP-RNAase. The projection is of the difference density between sections 0.33 through 0.37 along the c axis on the n-b plane of the unit cell of the crystal. Location of some of the residues in this region are marked. This map was made with data to about 3 A resolution and involved 2374 reflections. Heavy lines and large X’s show course of peptide backbone and acarbons, respectively. Light line with small x’s shows side chain of Lys-41. X’s in circles show the heaviest densities of the side chains of Tyr-73 and -1 15, Arg-10, Pro-114, Glu-11, and the phosphate ion (large circled X). The heaviest density difference, at the terminus of Lys41, is assigned to the CDNPgroup. The positive and negative densities near the phosphate are probably due to a small shift of the phosphate. Right: A projection of the electron density of the native protein between 0.33-0.37 in c. This map is computed at 2 A resolution using 8189 reflections. The side chain of Lys-41 is indicated. The high density in the upper right is the 40-95 disulfide bridge. Contour levels in this map are about 10 times higher than in the difference density map.

208

ARYLATION OF RIBONUCLEASE

Lys-41 with TNBS was estimated to be 300 times that of Lys-I. In that work, it was not demonstrated chemically that Lys-41 was the very reactive site, but was assumed to be so on the basis of a kinetic analysis indicating one site to be much more reactive than the others. In the second report (Goldfarb et al., 1974), in which the modified residues were identified through isolation of the peptides, the amount of modified Lys41 was only twice that of Lys-1. This is not incompatible with Lys-41 being the most reactive group since in the second work the ratio TNBS: RNAase was 400. At this high ratio the rate of modification of Lys-41 presumably is limited by saturation of the site, and the large excess of TNBS modifies Lys-1 at a comparable rate. It is a common practice to carry out chemical modification at high reagent :protein ratios, and to quench the reaction when the fraction of unmodified protein molecules (or remaining activity) has fallen to a predetermined extent. If it is desired to obtain a high degree of specificity of site of reaction, it is advisable to use a low ratio of reagent:protein. On the other hand, if it is desired to find out if several moderately reactive sites exist it is advisable to use a larger ratio of reagent-to-protein.

Preferential reaction at lysyl-41 The pK of Lys-41 appears to be lower than normal for ane-amino group, about 8.8, accord- ing to results from 'Hn.m.r. (Brown & Bradbury, 1975, 1976). From reactivity toward TNBS Goldfarb et al. (1974) concluded that the pK is about 9. Murdock et al. (1976) studied the pHdependence of the reactivity of Lys41 toward FDNB, and concluded that it was governed by a group with pK,, of 8.8. A pK of 9.1 was derived by Carty & Hirs (19683) from the reactivity of RNAase with SNFB; from their Figs. 7 and 8, pK values up to 9.5 may be inferred depending on the pH range, as noted by these authors. Goldfarb et al. calculated pK, values of 7.9-8.3 for six other a-amino groups based on the pHdependence of reaction with TNBS. (The pK values of the other three were not investigated.) These values are much below the average pK obtained by titration (Tanford & Hauenstein, 1956). Although Goldfarb obtained by the chemical reactivity method pK

values for Na-acetyllysine, glycine and glycyl- glycine in agreement with titration values, for proteins the method can give pK values having a meaning different from those obtained by tit;ation. The low pK values found by Goldfarb et al. (1974) for the €-amino groups other than Lys-41 might be the artifactual result of using data only from the low pH limb of a bell-shaped curve, which could result from the participation of other titratable groups. But Carty & Hirs (1 368b) using SNFB obtained an average pK of 10.1 for the nine other €-amino groups, a value close to the potentiometric value of Tanford & Hauenstein (1956). The reason for the diver- gence of results is not obvious.

A low pK would result in greater reactivity. Orientation factors may also be involved. Specific orientation of a reagent has been proposed for the rapid carboxyniethylation of the active site histidyl side chains of RNAase by haloacetates (Crestfield et al., 1963; Heinrikson, 1966; Fruchter & Crestfield, 1967). However, the similar AS* values for carboxymethylation of the active-site histidyl residues of RNAase and of histidyl hydantoin puts this interpretation into question; enthalpic factors may be more important (Lennette & Plapp, 1978).

The selectivity of CDNCB toward RNAase appears to depend on a combination of sluggish reactions with unactivated amino groups, and a moderately facilitated reaction in the active site. The pH-independent rate constants are 0.66 M -' min-' for Lys-41 and 0.08 M-' min-' for Na- acetyllysine, a ratio of 8 : 1 based on pK 10.8 for Na-acetyllysine (Goldfarb, 1974) and 9.0 for Lys-41. Since an incipient chloride ion is formed in the transition state, positive charge in the site is expected to facilitate the reaction. This could account for the favourable AH* of carboxymethylation of RNAase compared with histidyl hydantoin.

A similar consideration may be applicable to the hydrolysis of cyclic nucleotides by RNAase. The positively charged active site of RNAase will not only facilitate binding of the substrate, but may bind the product more strongly, since the latter is a doubly charged phosphate monoester and the substrate is singly charged. Thus, a cationic active site will promote the departure of the leaving group from the 2'- position of a cyclic nucleotide.

209

J . BELL0 ET AL.

The E-amino of Lys-41 is also more reactive toward CDNCB at pH 8.2 than is the a-amino of Lys-1, which has a pK of 7.8 (Tanford & Hauenstein, 1956). The a-amino is a weaker nucleophile than is the e-amino of Lys41, probably from the same cause as its low pK, the inductive effect of the peptide group. By contrast, if Lys41 has a low pK, this is the result of nearby positive charges which would not weaken the nucleophilic character of Lys-41.

A low pK of Lys-41 could account, in part, for its high reactivity toward other reagents, e.g. pyndoxal phosphate (Riquelme et al., 1975; Raetz & Auld, 1972), haloacetates and halo- acetamides (Heinrikson, 1966). Both active site lysyl residues, Lys-7 and Lys-41, react readily with pyridoxal phosphate, the ratio of reactions at the two sites being dependent on the conditions of buffer and pH (Means & Feeney, 1971; Raetz & Auld, 1972, Riquelme et aL, 1975). There is one common class of reagent with which Lys-41 is the least reactive lysyl residue, namely that of the guanidinating agents 0-methyliso-urea and guanyl-3 ,5- dimethylpyrazole (Brown & Bradbury, 1976; Click & Barnard, 1970) and amidinating agents (Hartman & Wold, 1967). This probably results from these reagents reacting in the protonated form. Positive charges in or near the active site would inhibit these reagents.

In other work from this laboratory (Iijima et al., 1977) it was shown that when Arg-39 and -85 are modified by a ketoaldehyde, the reactivity toward CDNCB is decreased to 25% of that of native RNAase at pH8 and 3% at pH 7. Carty & Hirs (1968b) had suggested that since the anomalous pK behavior of Lys-41 persists to pH 10, an arginine residue influences Lys-41. Our results on kethoxal-modified RNAase are compatible with this idea. Since arginyl residues 39 and 85 are not in the active site cleft it is unlikely that the reduced activity toward CDNCB is a result of steric hindrance of Lys41. Modification of arginine lowers the pK of the guanidino group to about 6 (Iijima et al., 1977). The loss of two arginyl positive charges could raise the pK of Lys41, resulting in a decrease in reactivity of Lys-41. The decrease in reactivity at pH7 and 8 is compatible with a pK increase from 8.8 to about 9.5-10. Reac-

tivity of Lys-41 toward CDNCB presumably requires an unprotonated amino group, since such reactions proceed more rapidly at higher pH. We had noted earlier (Iijima et al., 1977) that modification of Arg-85 mght break its interaction with Asp-83, with loss of some conformational stability. This could result in a decrease of reactivity toward CDNCB. Loss of native conformation often makes functional groups more reactive through greater accessi- bility. But when, in the native protein, a group is in an activating environment, loss of native conformation may result in a decrease in reactivity (Stark et al., 1961).

The X-ray structure of crystalline RNAase shows the following cationic residues in or near the active site: Lys-1, Lys-7, Lys-66, Arg-10, Arg-33, Arg-85, His-12 and His-119. Arginyl residues 10, 33 and 85 are neutralized by hydrogen bonds to (3111-2, Asp-14 and Asp-83, respectively. Arg-39 is often considered to be near Lys-41, being only two residues distant in the primary sequence; the X-ray structure shows the guanidino group to be distant. In solution, rotations around side chain bonds could bring it closer. but this would require an approach against a repulsive charge gradient (which would be decreased by an anionic ligand). Above pH 7.5 all of the histidyl side chains are uncharged, leaving essentially two nearby (6-8 A) cationic residues (Lys-7 and Lys-66) and two more distant groups, Lys-37 and Arg-39 (about 10 A), not compensated by closely associated carboxylate groups, to influence the pK of Lys-41. Possibly, the eamino group of Lys-1 may also have an influence. This side chain cannot be seen because of large thermal movement. It should be noted that at any moderate ionic strength the electrostatic effect of external residues, such as Arg-39, will be much reduced. The same would likely be the case when a polymeric substrate is bound, since phosphodiester groups would likely be near the cationic side chains.

When an anionic ligand is bound in the active site the pK of Lys-41 would be expected to increase. Such an effect was observed by n.m.r. for the pK of N-methyl-Lys-41 in the presence of phosphate (Brown & Bradbury, 1975) and for the active site histidyl side chains when phosphate was bound to RNAase S (Cohen &

210

ARY LATION OF RIBONUCLEASE

Shindo, 1975) or 3'-CMP to RNAase (Meadows et al., 1969; Ruterjans & Witzel, 1969; Griffrn et al., 1973; Pate1 et aZ., 1975). (The n.m.r. spectra of the two histidyl residues not in the active site are not affected by phosphate (Brown & Bradbury, 1975).) Therefore, when CDNCB is bound in the active site cleft, the pK of Lys-41 may be higher than in the free protein. In conformity with this idea is the fact that the pK value of 9.1-9.5 for Lys-41 obtained from the reactivity toward anionic SNFB (Carty & Hirs, 1968b) is significantly greater than the pK of 8.8 from the reactivity toward uncharged FDNB (Murdock et al., 1966). The closer the pK is to the normal pK, the more important become other factors affecting reaction rates.

Crystallography The CDNP-RNAase crystal is very closely isomorphous with the native protein (see Experimental), more closely isomorphous than for the pair DNP-RNAase S and RNAase S (Allewell et al., 1973). The carboxylate group of CDNP may be responsible, having an effect similar to that of binding 3'-pyrimidine nucleo- tides to DNP-RNAase S (Mewell e l aZ., 1973). At 3 A resolution it is not possible to resolve the nitro and carboxyl groups so that the orientation of the carboxylate toward cationic sites is not known. The crystallographic data indicate that despite the presence of the bulky cationic CDNP group, the phosphate is not extruded from the active site, but only displaced by a samll distance. In native RNAase crystals, phosphate is bonded to Lys41 and to other sites. Presumably, in CDNP-RNAase the phos- phate remains bound to the other sites.

ACKNOWLEDGMENTS

We thank Dr. N.D. Potti for preparing CDNP-methyl- amine and CDNPglycine and for the preliminary studies with 6-carboxy-2,4dinitro-l,3dichloroben- zene, Dr. A. Grossberg and Mr. R. Chrzanowski for amino acid analyses, and MIS. T. Falzone for crystal- lizing CDNP-RNAase.

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