Mechanisms of Carboxymethylation Nudeases by Haloacetates and Tosylglycolate*

SUMMARY Certain histidine residues in bovine pancreatic ribonuclease and deoxyribonuclease react with iodoacetate about 1UOO times faster than free histidine reacts. The mechanisms of these facilitated, active-site-directed reactions were investi-gated by study of the kinetics of carboxymethylation with reagents with varied leaving groups: chloride, bromide, iodide, and tosylate. With ribonuclease, the rate of reaction gave hyperbolic dependence on the concentration of reagent. The data fit a Michaelis-Menten mechanism, with dissociation constants of 8 to 24 rnM for the various reagents. The similar magnitudes of the constants lead to the conclusion that the reagents bind (reversibly) in a common way to the enzyme. The pseudo bimolecular rate constants for reaction (after correction for the differing inherent reactivities of the reagents) were also similar, indicating that the leaving group is not very important in the mechanism. These studies support the proposal that the reaction of haloacetates with ribonuclease is facilitated by ionic- attraction of the carboxylate ion by a protonated imidazole

BRYCE V. PLAPP$ From The Rockefek Lkiversity, Nets York, New York 10021, and the Department uf Biochemistry, The Universit.y of IOWU, 10~ City, Iowa 52242 SUMMARY Certain histidine residues in bovine pancreatic ribonuclease and deoxyribonuclease react with iodoacetate about 1UOO times faster than free histidine reacts.
The mechanisms of these facilitated, active-site-directed reactions were investigated by study of the kinetics of carboxymethylation with reagents with varied leaving groups: chloride, bromide, iodide, and tosylate.
With ribonuclease, the rate of reaction gave hyperbolic dependence on the concentration of reagent.
The data fit a Michaelis-Menten mechanism, with dissociation constants of 8 to 24 rnM for the various reagents. The similar magnitudes of the constants lead to the conclusion that the reagents bind (reversibly) in a common way to the enzyme.
The pseudo bimolecular rate constants for reaction (after correction for the differing inherent reactivities of the reagents) were also similar, indicating that the leaving group is not very important in the mechanism.
These studies support the proposal that the reaction of haloacetates with ribonuclease is facilitated by ionic-attraction of the carboxylate ion by a protonated imidazole while an unprotonated imidazole displaces the halide ion. In contrast, the kinetics of carboxymethylation of the Cu2+-Tris complex of deoxyribonuclease showed that the reagents bound loosely, with dissociation constants of 130 xxl~ or larger.
Furthermore, the corrected, pseudo bimolecular rate constants increased as the size of the leaving group decreased.
The larger groups may be more sterically hindered in the reaction; alternatively, the smaller groups may be attracted more to the electrophilic Cu2+ to show that bovine pancreatic ribonuclease has 2 neighboring histidine residues (12 and 119) that are involved in the mechanism of action of the protein.
The success of these studies depended upon the fact that the rea,ction of iodoacet'ate with these histidine residues was about 1000 times faster than the reaction with histidine free in solut,ion under the same conditions (3). The facilitation of the reaction had been explained by postulating that the prot,onat,ed imidazole nit#rogen of one histidine attracted the carboxylate anion of iodoaceta-te, orienting the reagent for attack by the unprotona,ted nitrogen of the other imidaaole (4)* The pH dependence observed for the reaction (5-7), the fact that iodoacetamide reacts much more slowly than iodoacetate (4), the mutua,lly exclusive alkylation of one histidine or the other (2), and t'he three-dimensional structure determined by x-ray crystallography (8, 9) fit this explanation.
Price et nl. (10) showed that iodoacet,ate, but not iodoac&amide, inactivated bovine pancrea,tic deoxyribonuclease by ca,rboxymethvlating 1 histidine residue. A divalent metal ion was required for the reaction; with Cu 2+, the rate of inactivation was about 1000 times faster than the rate of reaction of free histidine with iodoacetate.
The facilitation was explained by postulating tha,t the metal cation attracted the iodoacet8ate anion, so that a neighboring imidazole group could be alkylated.
The mechanisms proposed for the carboxymethylation of the nucleases presume Michaelis-Ment'en behavior, in which a, noncovalent, complex of enzyme and iodoacetate forms and is converted to t,he irreversibly modified enzyme: The noncovalent, complex has not been directly detected, nor has its dissociation constant (K = kz/kI) been determined.
We have studied the kinetics of carboxymethylation in order to confirm t'he mechanism and to determine the kinetic constan& Iodo-, bromo-, and chloroacetates and tosylglycolate (carboxvv 4897 methyl to&ate) were used to study the effect of the leaving group (halide ion or tosylate) on the reaction.
The p-toluenesulfonyl group (tosyl) is used T\-idely in synthetic and physical organic chemistry as a leaving group; it is displaced by nucleophiles more easily than iodide (11). We thought that Cosylglycolate might have some affinity for the active sites of RNase and DNase or might have some special reactivity because of the similarity of the leaving anion t,o the phosphodiester substrates.
Examination of a three-dimensional model of RNase S (9) showed that the tosyl group could fit into the cleft which holds the pyrimidine base of the substrate, near phenylalanine 120 and valine 43, while the carboxylate was attracted to lysine 7 or 41 and the methylene carbon was attacked by N-l of hist'idine 119.

EXPERIMENTAL PROCEDURE
Materials-Chloroacetic acid (zone-refined), bromoacetic acid, and iodoacetic acid were obtained from Aldrich. Tosylglycolate was prepared by the two-step procedure of Lichtenberger and Faure (12)  Na(X (1.96 g, 0.04 mole) was dissolved in 5 ml of I-120 and cooled to 0". Formaldehyde (3.05 ml of 37% CH20, 0.04 mole) \vas added dropwise with stirring over a period of 15 min. Recrystallized p-toluenesulfonyl chloride (5.7 g, 0.03 mole) was added over 20 min and the cloudy white mixture was stirred at 0" for 1 hour and allowed to warm up to room temperature for 30 min. The white crystals were collected by filt'ration, washed with water, and dried in vacua for 2 hours; yield was 5.7 g. The crystals were dissolved in 50 ml of diethyl ether and the solution was dried over CaC12, filtered, and evaporated to a small volume under vacuum.
A second crop of crystals was collected upon further concentration: 2.2 g, m.p. < 35". The crude tosylglyconitrile (second crop) was dissolved in 55 ml of 0.2 31 NaOH.
The mixture was stirred for 50 min at room temperature and filtered, and the filtrate was acidified to pH 1.2 with concentrated HCI. The product that formed (0.45 g) was collected by filt,ration and recrystallized by dissolving it in 25 ml of 0.2 hf NaOH and acidifying the solution again to pH 1.2 with concentrated HCl. The crystals were dried over CaS04 in uacuo, yield 0.3 g (4'%) : m.p. 132-135" (literature 137"). In the synthesis described, 14C can be introduced into t'he carboxyl group of the reagent. Eonradioactive reagent is prepared in better yields from commercial cyanomethyl p-toluenesulfonate (Aldrich).
The reagent has been stored for over 2 years in a desiccator without change in melt,ing point or reactivity.
Bovine pancreatic RYase A (phosphate-free) and DNase (DP grade), yeast RNA, and calf thgmus DNA were purchased from TYorthington.
The A form of DNase (13) was used in the following studies.

Carboxymethylated
RNasc was hydrolyzed (17) and analyzed for amino acids (18) with accelerated systems (19) on columns of Beckman-Spinco h-k-15 (0.9 x 55 cm) and AA-27 (0.9 x 11 cm). The color value of l-carbosymethylhistidine was taken as that of glycine (2). The color value of cysteine was taken as 0.15 times t'hat of leucinc (20) and that of X-carboxymcthylcysteine as 0.93 times that of aspartic acid (18). llkaline hydrolysis and ninhydrin analysis were also performed (21,22).
The kinetics of inactivation of RNase by various carboxymethylating reagents was studied at 37" with 0.20 M sodium 2-(Wmorpholino)ethanesulfonatc buffer, pH 5.50. This buffer was chosen because it did not affect the rate of inhibition by iodoacetate; sodium acetate buffers mere strongly inhibitory. Reactions with iodoacetate were protected from light. The data were fitted to the equation for all hyperbola, by means of a least squares method and on the assumption of equal variance of the velocities (23).
The carboxymethylation of D1Casc was studied wit,h reaction mixtures of 0.4 ml final vohmie prepared as follo\vs. To 0.1 ml of 4 mg per ml of dialyzed and lyophilizcd DNase iz. suspended in water was added 0.1 ml of 16 ml{ CuCl:! in 0.1 M Tris-HCl buffer, pH 7.2, that was readjusted to p1-l 7.2 with 1 M NaOH; 0 to 0.2 ml of 0.05 M Tris-HCl buffer, pH 7.2, was added, and the reaction was initiat'cd bv the addition of 0 to 0.2 ml of 0.2 M reagent dissolved in 0.2 11 NaOH and 0.05 M Tris-HCl buffer, pH 7.2. The final pH was 7.2 and the temperature of the solutions and reaction mixtures was 25".
The relative chemical reactivity of tosylglycolate and the haloacetates was determinrd by the reaction with 4-(p-nitrobenzyl)pyridine (24) according to the procedure of Baker and Jordaan (25).

Reactivity of Tosylglycolafe-To
show that tosylglycolate II-as a carboxymethylating reagent, we st'udied its reaction with cysteine.
The pH was maintained between 8 and 10 for 2 hours at room temperature, and the reaction was stopped by acidification to pH 1.2 with HCl.
The solution was made up to 10 ml with HZ0 and diluted 20.fold with pH 2.2 sample buffer. A 45cY0 yield of carbosymethylcysteine was obtained as well as 25c ,,( cysteine and 27% half-cystiane.
A 3% yield of a; unknown compound at the threonine position was also seen, but this could have been a contaminant in the cysteine.
RNase was slowly inactivated by tosylglycolate. The site of alkylation was determined b)-chromatographic analysis of the products of the reaction (Fig. 1) alld by amino acid analysis. The major product from the tosylglycolate reaction corresponded in elution position to the major product from the iodoacetate reaction, bhat is l-carboxymethylhistidine 119.RNase (26). A small amount of 3-carboxymethylhistidine 12.RNase (rlutcd at about 185 ml) was also present.
(Some RXase iz and some minor, active peaks eluted before the inactive derivatives) The ratio of histidine 119 to h&dine 12 products was about 17 : 1, but the ratio could not be determined accurately because the histidine 12 derivative was present in such small amounts.
For the iodoacetate reaction the ratio was about 7:1, which agrees with the ratio found previously at 25" (21). Thus tosylglycolate reacts with somewhat more preference for histidine 119 than does iodoacetate.
The major product from the tosylglycolat'e reaction was desalted by gel filtration on a column of Sephadex G-25 (medium) A, RNase A (phosphate free), 20 mg, in 2 ml of 0.1 M sodium acetate buffer, pH 5.5, was treated with sodium tosylglycolate (36 pmoles) for 10 hours at room temperature and 12 hours at 37", after which time the enzyme had lost 90(?; of its activity. The products were chromatographed on a column (0.9 X 57 cm) of sulfoethyl-Sephadex C-25 [fine (2.0 meq per g)] equilibrated with 0.1 M sodium phosphate buffer containing 0.055; phenol, pH 6.60, at 25" with a flow rate of 10 ml per hour (26). B, RNase A, 11 mg, in 1.0 ml 0.1 M sodium acetate buffer, pH 5.5, was treated with sodium iodoacetate (36 qnoles) at 25" for 195 min, at which time the enzyme had lost 600/, of its activity, and the products were chromatographed as in A. The peaks were located by analysis with ninhydrin and identified from their elution positions (26 (2 X 40 cm) equilibrated with 5% acetic acid, lyophilized, and hydrolyzed to the amino acids. As compared to the composition of RNase A, there was one less histidine, and 0.9 residue of lcarboxymethylhistidine was found. Kinetics 05 Carboxymethylation-When RNase was treated with more than a 100.fold molar excess of tosylglycolate, the inactivation followed pseudo first order kinetics (Fig. 2). Similarly, inactivation by bromoacetate, iodoacetate, and chloroacetate was pseudo first order.
With all four reagents, however, as the concentration of reagent Ivas increased the rate of inactiva- tion increased, but not with first order dependence.
The increase was hyperbolic, which is readily apparent from the double reciprocal plots (Fig. 3). The kinetic constants derived from such data are given in Table I. The relatively small standard errors leave little doubt that the data fit hyperbolic rather than first order kinetics.
The inactivation of DNase by carboxymethylating reagents was pseudo first order with respect to DNase activity, as shown previously (10). As with RNase, the rate of inactivation of DNase by tosylglycolate, bromoacetate, and iodoacetate gave hyperbolic dependence on the concentration of reagent, as shown by the double reciprocal plots (Fig. 4). The inactivation by chloroacetate, however, was first, order with respect to its concentration (Fig. 4B). The kinetic constants for carboxymethylation of DNase are given in Table I.
In an experiment to test the effect of another metal ion, the enzyme was treated with 25 mM iodoacetate in the presence of 10 or 50 mM CdC&, at 25" and pH 7.2. The calculated second order rate constant was 0.14 M-I m&l. DISCUSSIOK The hyperbolic dependence of the rate of inactivation of RNase on the concentration of carboxymethylating reagent supports the proposal (4) that the reagents bind reversibly to the enzyme before reacting chemically.
Such kinetic behavior is expected for active-site-directed reagents (27,28). The affinity of the reagent for the active site has been used to explain the faster rate of reaction of amino acid residues in the enzyme as compared to free amino acids. Since RNase binds the reagents relatively weakly, the precise orientation of the bound reagent apparenbly facilitates the chemical reaction.
Dafforn and Koshland (29) have predicted that a properly oriented intramolecular (or en- reaction may occur up to lo4 times faster than the comparable bimolecular reaction. At pH 5.5 and 25", the second order rate constant for the reaction of free histidine and bromoacetate is 6.2 x 10M4 11-l mine1 (3), and the pseudo bimolecular rate constant (kJK) for the reaction of RNase and bromoacet,ate at 25" is 4.6 11-l min-l.
Thus RNase facilitates the reaction 7400-fold, and it appears that RNase may bind and react with bromoacetat,e as if the reagent were a substrate for the enzyme.
Therefore, st'udy of the mechanism of carboxymethylation of RNase may lead to better understanding of the mechanism by which RNase hydrolyzes RNA.
Furthermore, the pK values for the histidines in the free enzyme and its haloacetate complexes can be determined from the pH dependence of carboxymethylation.
The similar magnitudes of K for RNase in Table I indicate that the leaving group is not very important in the binding of reagent and lead to the conclusion that the four reagents bind in a common way to the enzyme.
Presumably the carboxymethyl group fits between the two imidazole groups. The magnitudes of Jc3 in Table I do differ greatly, but, of course, the chemical reactivities of the reagents differ also. In order to compare the over-all reactivities of the reagents with RNase, the pseudo bimolecular rate constants (As/K) were simply divided by the relative reactivities with 4-(p-nitrobenzyl)pyridine.
(The relative rates of reaction of different nitrogen nucleophiles with reagents with halide or tosylate leaving groups are similar (30).) Although the reagents \T-ith the smaller leaving groups, chloride and 4899 bromide, may be somewhat more reactive than the reagents with larger leaving groups, the differences are not very large. It appears, rather, that the leaving group is not very important in the facilitation of carboxymethylation.
Since the leaving groups differ greatly in solvation energies, polarizability, and size, it seems that the enzyme interacts with the leaving groups in a common way or, more likely, not at all during the carboxymethylation.
As with RNase, the reaction of a histidine residue in DNase with iodoacetate is greatly facilitated.
In contrast to RNase, DNase binds the reagents loosely, with dissociation constants larger than 0.1 M. If the affinity of the reactants actually facilitates the reaction, the orientation of reactants in the DNaseinhibitor complex must be more favorable for reaction than in the RNase-inhibitor complex. However, with DNase, the magnitude of the facilitation (k/K COW, in Table I) increases as the size of the leaving group decreases. Steric hindrance could decrease the rates of reaction of tosylglycolate and iodoacetate as compared to chloro-and bromoacetates.
On the other hand, DNase may have a (catalytic) group that facilitates the formation of the transition state by interacting with the leaving group. Since a divalent metal ion is required for carboxymethylation, the ion could act as an electrophilic catalyst.
The relative rates of reaction, chloro > bromo > iodo, and CL@ > Cd2+ >> Mn2f > Ca2+ (lo), are consistent with the stabilities expected for complexes of the metal halides (31). It should be noted that a variety of divalent metal ions, including Cu2+, activate Dn'ase for hydrolysis of DNA (32, 33). The mechanism of carboxymethylation of DNase may involve ionic attraction of the carboxylate ion and electrophilic catalysis by Cu2+. Comparison of the carboxymethylation of Rn'ase and DNase indicates that these enzymes facilitate (catalyze) active-site-directed reactions by orienting the reactants or by using catalytic groups or both.
Tosplglycolate carbosymethylates sulfhydryl and imidazole groups and is about one-eighth as reactive as iodoacetate with a nucleophilic nitrogen base. Tosyl&colate is not specially reactive with RNase and has no more affinity for the active site than do haloacetates.
It reacts with histidines 119 and 12 with a ratio of about 17: 1, and thus is more selective for histidine 119 than are the haloacetates, but less selective than some long chain a-bromo acids (3). The reagent may be used to carboxgmethylate proteins that react too fast with iodo-or bromoacetate and too slowly with chloroacetatc, or where the halide ions interfere in the reaction.
Alternatively, it may be used xl-here the affinity of the reagent for the active site may be a significant, factor in obtaining: a specific reaction.
While this work was in progress, Sakagawa and Render (34) used methyl p-nitrobenzenesulfonatc to methylate histidine 57 in chymotrypsin.
Since tosylates are readily prepared from alcohols, active-site-directed reagents of many compounds can be made.