reaction of the catalytic subunit of escherichia coli .aspartate

THE JOURNAL OF T~IOLOGICAL CHEMISTRY Vol. 240, No. 10, Issue of May 25, pp. 3151-3159, 1971

Printed in U.S.A.

Reaction of the Catalytic Subunit of Escherichia coli .Aspartate Transcarbamylase with Permanganate Ion, a Reactive Structural Analogue of Phosphate Ion*

(Received for publication, November 20, 1970)

WILLIAM F. BENISEK

From the Department of Biological Chemistry, School of Medicine, University of California, Davis, California 95616,$ and The Department of Biochemistry, Stanford University, Stanford, California 94305

SUMMARY

A reaction between the catalytic subunit of Escherichia coli aspartate transcarbamylase and potassium permanganate is described. The reaction is accompanied by a rapid loss of the enzyme’s catalytic activity even when stoichiometric amounts of permanganate and enzyme are ‘used. Charac- terization of the inactivated enzyme has shown that when stoichiometric amounts of permanganate are used for the inactivation the only chemical change detected is oxidation of the enzyme’s three cysteine thiol groups to sulfonate groups. The rate of inactivation of the enzyme by permanganate was found to be greatly decreased by the substrate carbamyl phosphate and to a lesser extent by the inhibitors phosphate, pyrophosphate, and succinate. Competition experiments show that the thiol groups of the catalytic subunit are approximately 45 times as reactive toward permanganate as the thiol group of Z-mercaptoethanol. This result is to be contrasted to the results of Vanaman and Stark (.I. Biol. Chem., 245, 3565 (1970)) which showed that the thiol groups of catalytic subunit are hyporeactive or unreactive toward the many thiol reagents that they tested. The facil- ity with which permanganate reacts with the protein’s thiol groups may be a result of the structural resemblance between permanganate and the competitive inhibitor phosphate ion, a resemblance which is not shared by the other thiol reagents. Certain chemical properties of permanganate ion are pointed out which recommend it as a potential affinity reagent for phosphate binding sites in other proteins.

The enzyme aspartate transcarbamylase of Escherichia co& catalyzes the transfer of a carbamyl group from carbamyl phosphate to the amino group of aspartic acid in the first committed step in the biosynthesis of pyrimidine nucleoside triphosphates

* This work was supported by Grant GM 11788 from the Na- tional Institutes of Health (to George R. Stark) and by a Uni- versitv of California Facultv Research Grant (to William F. Benisek).

1 Communications regarding this publication should be sent to this address.

in this organism (I). The regulatory role that this enzyme plays in this biosynthetic pathway has been well established (2) and has been the object of continuing study in a number of laboratories. Less attention has been paid to the catalytic mechanism itself, but recently a series of papers by Stark and co-workers (3-5) has appeared which describe studies of the mechanism of catalysis by the isolated catalytic subunit (6), a fragment of the native enzyme which is devoid of the native enzyme’s regulatory properties but which efficiently catalyzes the transcar- bamylation reaction.

The mechanistic picture which has emerged from these studies can briefly be summarized as follows. The enzyme first binds carbamyl phosphate, and then aspartate, to form a ternary complex. The binding of aspartate is thought, on the basis of ultraviolet difference spectra and steady state kinetic, nuclear magnetic, and other measurements to be accompanied by a change in the conformation of the enzyme. Transfer of the carbamyl group occurs in the ternary complex to produce a new ternary complex involving the enzyme and the products carbamyl aspartate and phosphate ion which dissociate in that order from the active site.

Structural studies by Weber (7) have revealed that the native enzyme is a complex composed of six copies of each of two kinds of polypeptide chains, regulatory chains, R (mol wt 17,000), and catalytic chains, C (mol wt 33,000). Treatment with p-hy- droxymercuribenzoate produces fission of this structure into two species, regulatory subunits which contain two R chains and catalytic subunits which contain three C chains. Thus the molecular weight of catalytic subunit is approximately 100, 000. Earlier studies by Weber (8) and Her& and Stark (9), have indicated that the polypeptide chains of catalytic subunit are very similar if not identical.

The functional groups of catalytic subunit involved in the binding of substrates and the catalysis of their reaction are not known. This report describes experiments designed to detect the proximity of particular functional groups in the binding and active sites of this enzyme by the approach of affinity labeling (10). The experiments described in this report represent an exploration of the use (of a structural analogue of the product and competitive inhibitor, nhosphate ion, as a reagent for specific modification of functional groups in or near the phosphate binding site of catalytic subunit. The phosphate

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3152 Reaction of Aspartate Transcarbamylase Catalytic Xubunit with Permanganate Vol. 246, No. 10

analogue chosen is permanganate ion. Like phosphate, it possesses over-all tetrahedral symmetry and a manganese- oxygen bond length which is close to the phosphorus-oxygen bond length of phosphate ion (11, 12). In the neutral pH range phosphate carries one to two negative charges depending on the pH, whereas permanganate possesses one negative charge over the entire pH range of interest in biochemical work. These structural and electrical similarities suggested permanganate as a candidate for study.

The notable difference between phosphate and permanganate which forms the basis for affinity labeling is that permanganate, unlike phosphate, is an extremely reactive and powerful oxidizing agent. Most functional groups in organic molecules are potential substrates for oxidation by permanganate although wide differences exist in the reactivity of various organic structures toward this reagent. At first, this extremely broad specificity of permanganate as an oxidizing agent would seem to be a dis- advantage in chemical modification work with such complex molecules as proteins. However, the broad specificity of this reagent ensures that there is relatively little restriction on the kinds of functional group which must be present in a phosphate binding site of a protein in order to have reaction with the reagent. Since permanganate can oxidize alcohols, amines, phe- nols, indoles, imidazoles, thiols, disulfides, alkenes, and even saturated hydrocarbons (13), it is clear that the use of permanganate as a reactive probe of phosphate binding sites may be of some general interest and applicability.

This paper reports the reaction of catalytic subunit with very dilut,e solutions of potassium permanganate and characterization of the reaction in terms of the chemical processes occurring, its specificity, and some effects of substrates and inhibitors. A comparison is made between the reactivity of the enzyme toward permanganate and other chemical reagents.

MATERIALS AND METHODS

Enzylme--E. coli aspartate transcarbamylase and its catalytic subunit were prepared as described by Gerhart and Holoubek (6). Catalytic subunit had a specific activity of 0.5 to 0.6 mole of carbamyl aspartate formed per min per g of protein.

When examined in the Beckman model E analytical ultra- centrifuge, catalytic subunit sedimented as a single symmetrical peak (schlieren optics or ultraviolet scan system) with an x ohs of 5.28. Analytical disc gel electrophoresis in 0.017 M

sodium cacodylate (pH 6.5), 0.165 M Tris-HCl (pH 8.9), and 0.083 M Tris-borate (pH 9.4), in 5% polyacrylamide gels showed only single bands which were stained with Coomassie brilliant blue at each pH.

Catalytic subunit was stored at 24” in 0.04 M potassium phosphate, 2 mM 2-mercaptoethanol, 0.2 mM disodium EDTA, pH 7.0, at a protein concentration of 30 to 60 mg per ml. When stored under these conditions the specific activity remained unchanged over at least 1 year. Since 2-mercaptoethanol and EDTA both react rapidly with permanganate, it was necessary to transfer catalytic subunit to a different solution prior to its exposure to permanganate. This was done by dialysis of appropriately diluted enzyme against a suitable solution such as 0.025 M NaHCOa, pH 8.4 (in some early experiments), or against 0.01 M imidazole acetate, pH 7.0, while bubbling nitrogen through the solution outside the bag. In order to minimize a slow decrease in the thiol content and specific activity of catalytic subunit when stored in solutions free of 2-mercaptoethanol

and EDTA, such solutions were stored at 2-4” under a nitrogen atmosphere and were prepared within a few days of their use.

Reagents-Imidazole and mercaptoethanol were obtained from Eastman. Potassium permanganate was a reagent grade product of Mallinckrodt. Reduced glutathione, A grade, and glycylglycine, A grade, were purchased from Calbiochem. Guani- dine hydrochloride, Sequenal grade, was obtained from the Pierce Chemical Company. n-14C-Aspartic acid, 208 C per mole, was purchased from Schwarz BioResearch. For use in the enzyme assay it was diluted with n-aspartic acid, A grade, from Calbiochem. Dilithium carbamyl phosphate was a product of Sigma and was used without further purification. Sodium tetrathionate from K and K Laboratories was recrystallized from 50% aqueous ethanol.

Enzyme Assay-Catalytic subunit was assayed by the method of Porter, Modebe, and Stark (3). Reaction mixtures, 0.5 ml, contained 0.04 RI 14C-x-aspartate (-1 x lOlo dpm per mole), 0.01 M dilithium carbamyl phosphate, 0.2 M sodium glycylglycine, pH 8.0. The concentration of catalytic subunit was 2 to 50 pg per ml. The temperature was maintained at 28” by means of a continuously stirred, thermostated water bath. Aliquots, 100 ~1, were withdrawn at 30.see intervals with an Eppendorf automatic pipette. Generally, four time points were taken. A given sample of enzyme was assayed in duplicate or triplicate.

Protein Concentration-The protein concentration of un- modified and permanganate-modified catalytic subunit was determined by measurement of the absorbance at 280 mp. Based on protein concentrations determined by amino acid analysis, the absorbance of a 1 mg per ml solution of either of these species was 0.70 in 0.05 M glycylglycine, pH 8.0, or 0.01 M imidazole-acetate, pH 7.0. When solutions contained other species which absorbed strongly at 280 rnp, protein concentration was determined by amino acid analysis of acid hydrolysates.

Amino Acid Analysis--1cid hydrolyses were performed as recommended by Moore and Stein (14). Blkaline hydrolyses for determinations of methionine sulfoxide and tryptophan were conducted by a method’ with 5 N NaOH in the presence of starch. A starch-NaOH solution was prepared by dissolving 500 mg of Connaught partially hydrolyzed starch in 10 ml of 10 N NaOH which was maintained at 100” by a boiling water bath. Of this solution, 0.5 ml, while hot, was added to 0.5 ml of protein in water contained in a heavy walled Pyrex tube. The contents of the tube were frozen in Dry Ice-acetone and the tube was evacuated to less than 100 p Hg pressure and sealed without thawing the contents. Hydrolysis was carried out at 110” for 17 hours. Bfter the hydrolysis period the tubes were opened and the contents were acidified by addition of 1.0 ml of 5.4 N

HCl. This solution, containing precipitated silicic acid, was adjusted to an appropriate volume with 0.2 M sodium citrate buffer, pH 2.2. The silicic acid was removed by centrifugation and the supernatant solution was stored frozen until analyzed.

Half-cystine residues were determined as aminoethyl cysteine after alkylation of protein with ethyleneimine in 8 M guani- dinium hydrochloride, 0.01 M 2-mercaptoethanol, under a nitrogen atmosphere essentially as described by Cole (15). After acid hydrolysis of the ethyleneimine-treated proteins, analysis for aminoethyl cysteine was performed by using the

1 G. R. Stark and S. Moore, unpublished observations.


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Issue of May 25, 1971 W. F. Benisek 3153

columu, 0.9 x 55 cm, of the amino acid analyzer which had been equilibrated for 5 hours with 0.35 R’ sodiulll (citrate) buffer, pH 5.30. The color constant for aminoethyl cysteine was assumed to be identical with that of lysine.

Analyses of acid and alkaline hydrolysates were performed by using Beckman model 120B and 121 amino acid analyzers. Analyses were generally carried out in duplicate or triplicate. The areas under the chromatographic peaks were measured by means of a Beckman model 125 integrator. Small corrections were applied to compensate for errors in the integrator-selected base line.

Determirdon of Thiol Groups-Thiol groups were determined by a modification (16) of the method of Sedlak and Lindsay (17) with Ellman’s reagent, 5,5’-dithiobis(2-nitrobenzoic acid). Into small test tubes (8 x 75 mm) were introduced 150 ~1 of 8 M guanidine-HCl, 0.2 M Tris-HCl, 0.02 RI disodium EDTA (pH 8.2), 20 ~1 of 0.01 M DTNB2, 0.2 M Tris-HCl, 0.02 M disodium EDTA (pH 8.2), and 30 ~1 of a 1 to 5 mg per ml solution of protein in 2-mercaptoethanol-free buffer. The blank contained 30 ~1 of 0.2 M Tris-HCI, 0.02 M sodium EDTA (pH 8.2), in place of the protein solution. Along with each set of protein thiol determinations was run a standard composed of 150 ~1 of 8 M guanidine-HCI, 0.2 M Tris-HCl, 0.02 M disodium EDTA (pH X2), 20 ,uI of 0.01 M DTNB, 0.2 M Tris-HCl, 0.02 M disodium EDTA (pH 8.2), 20 ~1 of 1 mM reduced glutathione in water prepared within the hour, and 10 ~1 of 0.2 1\r Tris-HCl, 0.02 M

disodium EDTA (pH 8.2). The absorbance of all solutions was measured at 412 ml* against the blank. In each case, protein or glutathione was added last and the absorbance was measured within 30 sec. By using the known concentrat.ion of protein, the number of thiol groups per mole of catalytic subunit was calculated. Catalytic subunit which had been freshly dialyzed into mercaptoethanol-free buffer had a thiol content of 3.0 f 0.1 molts/100,000 g of protein. A standard curve obtained from determinations on increasing amounts of glutathionc was linear over a range of 0 to 0.1 mM glutathione.

Reaction of Catalytic Subunit with Potassium Perman.ganate-

To solutions of catalytic subunit (0.01 to 10 mg per ml) in 0.01 fir imidazole-acetate, pII 7.0, at 23-25” was added with stirring Q.01 rolume of aqueous KMnO~ containing 6 to 8 moles of KMn04 per mole of catalytic subunit to be reacted. After reaction for an appropriate time (a I mg per ml solution of catalytic subunit will completely react in a few seconds at 25’) the solution was rnade 0.01 M in 2-mercaptoethanol by addition of 1 M 2-mercaptoethanol. At this point aliquots may be taken for measurement of specific activity. I f it was desirable to remove excess 2-mercaptoethanol, the solution was dialyzed under nitrogen against 0.01 M imidazole-acetate, pH 7.0, or some other appropriate solution. The addition of 2-mercaptoethanol serves two purposes. First, it reduces any unreacted permanganate to manganous ion, thus terminating the reaction, and, second, colloidal manganese dioxide, a product of the reaction of permanganate with catalytic subunit, is also reduced to manganous ion. Thus all species of manganese are converted to the dialyzable species Mn++. At relatively high proteiu concentrations, the concentration of permanganate used is high enough to observe a very rapid disappearance of the pink color of permanganate ion.

In some cxperimcnts in which it was dcsircd to analyze reac-

2 The abbreviation used is: DTNB, 5,5’-dithiobis(2-nitrobenzoic acid).

tion mixtures immediately for thiol content without the intervcn- ing dialysis required to remove excess 2-mercaptoet,hanol, the reactions were terminated by addition of 1 M hydrazinc acetate, pH 7.0, to a final concentration of 0.05 M. Like mercaptoetha- 1101, this reducing agent converts all more highly osidized species of manganese to manganous ion.

RESULTS

Stoichiometry of Inactivation of Catalytic Subunit by Perman.ganate-In a preliminary investigation of the susceptibility of catalytic subunit to inactivation by KMn04, solutions of catalytic subunit in sodium bicarbonate solution were treated for 1 hour with quantities of permanganate ranging from 0.2 mole of MnO, per mole of catalytic subunit to 200 moles of lMnO,- per mole of catalytic subunit. As indicated in Table I, extensive inactivation of the enzyme occurred at a relatively low molar ratio of permangauate to catalytic subunit and at a very low concentration of permanganate, su ggesting that a rapid, specific reaction occurs between permanganate and catalyt’ic subunit which reduces the specific activity of the enzyme.

Since this reaction occurred at a significant rate (see below) at permanganate concentrations comparable to the concentration of catalytic subunit, it was possible to determine readily the stoichiometry of inactivation of catalytic subunit by permanganate. Fig. 1 summarizes the results of an experiment in which identical quantities of catalytic subunit were treated with varying amounts of KMn04 for a time sufficient for the reactions t,o come to completion. The resulting reaction mixtures were then assayed for enzymatic activity. This experiment, shows that the minimum quantity of permanganate required to inactivate catalytic subunit was 7.5 to 8 moles per mole of caata- lytic subunit. The portion of the curve between 0 and 4 moles of KMnO~ per mole of catalytic subunit was linear. Linear extrapolation of this portion of the curve produced a line which intersects the abscissa at 6 A= 0.5 moles of KhlnOa per mole of catalytic subunit. Apparently, permanganate reacts prefer- entially with a class of functional groups of catalytic subunit such that 6 moles of KMn04 are required for modification of these functional groups with consequent inactivation of the enzyme. At sufficiently great amounts of permanganate (7 to 8 moles per mole of catalytic subunit) some of the permanganate can react with a class of less reactive groups whose chemical modification by permanganate does llot inactivate the enzyme.

TARLE I Susceptibility of catall/tic subunit to extended treatment with

permanganate

Catalytic subunit (0.98 mg per ml; 9.8 MM) in 0.025 M NaHCOa was treated with varions amounts of KMnOa for 1 hour at 28”. At the end of this incubation period, aliquots of the reaction mixtures were diluted \T-ith 0.025 15 NaHC03 and assayed for enzymatic activity. No attempt, was made to reduce unreacted Mn04 or MnOs produced in the reaction.

Concentration of MnOF Molar ratio of MnOa- to catalytic subunit Specific activity

PX 0 2

20 200

2000

0 2.204 2.04

20.4 201

mole/?nin/g

0.481 0.471 0.271 0 0


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Reaction of Aspadate Transcarbamylase Catalytic Xubunit with Pelmanganate Vol. 246, No. 10

u 2 4 6 8 IO 12

MOLES KMnO,/MOLE CSU

FIG. 1. Stoichiometry of inactivation of catalytic subunit by KMnOa. Catalytic subunit used in this experiment was assayed for thiol content just before treatment with permanganate and was found to contain 2.99 equivalents of thiol groups per mole (lo5 g) of catalytic subunit. To a ZOO-4 portion of a 6.06 mg per ml solution of catalytic subunit in 0.01 M imidazole acetate, pH 7.0, was added 20 ,ul of aqueous KMnOa solutions containing 0 to 12 moles of KMn04 per mole of catalytic. subunit to be reacted. After incubation at 25” for 2 min any excess KMn04 and the product, MnOn, were reduced by addition of 20~1 of 0.1 M hydraeine sulfate, pH 7.0. Appropriately sized aliquots were diluted with 0.1 M glycylglycine-0.001 M mercaptoethanol, pH 8.0, prior to assay for enzymatic activity. CSU, catalytic subunit.

Catalytic subunit contains three similar if not identical sub-

units each of molecular weight 33 x lo3 (7). Hammes, Porter, and Wu (18) have recently found that there are three binding sites for carbamyl phosphate in each molecule of catalytic subunit. Assuming identity of these subunits we therefore can conclude that 2 moles of permanganate are required for inactivation of one subunit’s activity. Since reduction of Mn04- to MnOz requires 3 electrons, it is apparent that inactivation of each subunit is accompanied by abstraction of 6 electrons in oxidative processes involving permanganate. This conclusion rests on a number of assumptions. (a) Mn04- is functioning as a oxidizing agent. (b) The subunits of catalytic subunit are identical. (c) Inactivation of subunits does not affect the activity of the remaining subunits in the same complex.

Jlan.ganese Species Responsible for inactivation-The possi- bility existed that oxidation of functional groups by permanganate was not the chemical event which is directly responsible for inactivation of catalytic subunit but, rather, that some non- essential functional groups were oxidized by permanganate and that the resulting reduction products of permanganate, MnOs or Mn++, react with or interact with catalytic subunit in such a way as to result in inactivation of the enzyme. For example, the MnOz formed in the reaction is colloidal and can be centri- fuged at 27,000 X g for 10 min, yielding a brown pellet. Con- ceivably, inactivation of catalytic subunit could result from a nonspecific adsorption of the protein onto these colloidal parti- cles. h/ZnOz itself is a mild oxidizing agent and might be the species responsible for oxidative inactivation. Perhaps manganous ion can inactivate the enzyme by complex formation with potential ligand groups of the enzyme. Experiments were performed to examine these possibilities. Three experiments tested

1

Effect of reduclion products of permanganate on specijic activity oj cnlalylic subunit

In Experiment 1, 0.25 ml of 0.01 M imidazole acetate, pH 7.0, containing 0.04 M sodium fumarate was mixed wit’h 5 ,~l of 6 rnM KMnO1. Aft,er 1 min, 0.25 ml of 19.6 ,ULM solution of catalytic subunit in 0.01 M imidazole acetate, pII 7.0, was added and the mix- tllre was allowed to incubate for 1 min. The mixture was then diluted and assayed in duplicate. In Experiment 2? 0.25 ml of 19.6 PM catalytic subunit in 0.01 M imidazole acetate, pH 7.0, was mixed with 0.25 ml of 0.01 M imidazole acetate containing 0.04 M

sodium fumarate, pH 7.0. To the mixture were added 5 ~1 of 6 mM KMn04. After 1 min the solution was diluted and assayed in duplicate. In Experiment 3, 0.25 ml of 19.6 ,ULIM catalytic subunit in 0.01 M imidazole acetate, pH 7.0, was mixed wit,h 0.25 ml of 0.01 M imidazole acetate, pH 7.0. To this were added 5 ,uI of 6 mM KM1104 and the mixture was incubated for 60 sec. Then, it was diluted and assayed in duplicat)e. Experiment 4 was performed exactly as Experiment 3 except that an aliquot of the reaction mixture was treated with 0.001 M mercaptoethanol in 0.1 M glycyl glycine, pH 8.0, for 1 hour prior to assay. In Experiment 5, 0.25 ml of 19.6 pi~ catalytic subunit in 0.01 M imidazole acetate was mixed with 0.25 ml of 0.01 M imidazole acetate, pH 8.0. To this solution were added 5 .~l of 6 mM MnClz and t,he mixture was incubated for 1 min. Then an aliquot was diluted and assayed. Ex- periment 6 was the same as Experiment 3 but 5 ~1 of Hz0 were added instead of KMnOq.

Experiment and treatment Specific activity

molelmin/g

1. 60 mM freshly prepared Mn0~. 2. 60 PM MnO? prepared in situ.. 3. 6OpM KMn04.. 4. 60 ,UM KMnO4 + 1 mM mercaptoethanol. 5. 60 MM MnClz control. 6. Control.

0.376 0.375 0.011 0.012 0.387 0.387

the hypothesis that MnOz was the agent responsible for the inactivation. First, catalytic subunit was treated with freshly formed MnOz produced by reduction of permanganate with fumarate. Second, catalytic subunit was treated with permanganate in the presence of a sufficiently high concentration of fumarate so that most of the permanganate was reduced by fumarate rather than by the enzyme. Perha,ps MnOz thus produced in situ would be effective for inactivation whereas previously formed hInO would not. Third, a freshly prepared reaction mixture containing a permanganate-treated catalytic subunit and the MnOe which was produced by this treatment was exposed to mercaptoethanol, a reducing agent which reduces MnOz to Mn+f. Perhaps an MnOz-induced inactivation would be reversed by reducing the MnOz. A fourth experiment tested the susceptibility of catalytic subunit to inactivation by Mn++. The results of these experiments are given in Table II. They clearly show tha,t neither freshly prepared MnOz nor h!lnOz formed in the presence of catalytic subunit is effective in inacti- vating catalytic subunit. Also, enzyme which has been inactivated by permanganate is not reactivated by treatment with millimolar mercaptoethanol at pH 8, a procedure which rapidly reduces hlnOz to ;Vln++. Moreover, the specific activity of catalytic subunit is not reduced by treatment of the enzyme with manganous ion. Of the various forms of manganese tested only Mn04- was effective as an inactivator of the enzyme under


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these conditions. Thus, the inactivation of the enzyme by permanganate is t#he direct result of the rea.ction of permnnga- nate with functional groups of the enzyme.

Rate of Inactivation of Catalytic Subunit by Permanganate: Ef- fects of Substrates and Substrate Analogues-Initial attempts to measure the rate of inactivation of catalytic subunit by stoichiometric amounts of permanganate soon established that the reaction responsible for the inactivation was very rapid. How- ever, since stoichiometric amounts of permanganate could efficiently inactivate the enzyme, it was possible by working at

sufficiently low concentrations of catalytic subunit and per-

manganate, while maintaining a stoichiometric ratio of the two reactants, to reduce the rate of inactivation considerably. A

further minimization of the rate was achieved by carrying out

the reaction at 0”. Even so under these conditions the half-time

for inactivation of catalytic subunit by a B-fold molar excess of

permanganate was only 15 to 20 sec. Since the first aliquot of

the reacting mixture could not be taken sooner than about 15 set after start of the reaction, it was not possible to obtain very

accurate values for the initial rate of inactivation. However,

it was possible to observe at a semiquantitative level some effects of substrates and substrate analogues on the rate of inactivation

of catalytic subunit. These effects are shown in Fig. 2. These

results indicate that the reactivity of catalytio subunit toward permanganate is reduced when substrates which bind in the

80

60

0 20 40 60 80

TIME (SECONDS)

FIG. 2. Effects of substrates and substrate analogues on the rate of inactivation of catalytic subunit by KMn04. Of a 0.117 PIN solution of catalytic subunit in 0.01 M imidazole acetate, pH 7.0, 5.0 ml containing the indicated concentration of substrate or substrate analogue were treated with 0.6 mM KMn04 by rapid addition of 5 ~1 of 0.6 mM KMn04 with stirring. All reactions were at 0”. Just prior to starting the reaction a 500.~1 aliquot was removed for assay to determine the initial specific activity of the enzyme. After addition of the KMn04, further 500-~1 aliquots were taken at 15- to 20.set intervals and rapidly mixed with 5.~1 portions of 1 M 2-mercaptoethanol which had been previously placed in small test tubes. Generally four aliquots were taken over a period of 60 sec. The quenched reaction aliquots were then assayed for enzymatic activity without dilution. l , no additions; H, 0.001 M carbamyl phosphate; q , 0.001 M carbamyl phosphate plus 0.01 M succinate; 0, 0.01 M phosphate; a, 0.01 M

pyrophosphate; A, 0.01 M succinate.

active site of the enzyme are present. Such behavior is con- sistent with the presumption that whatever functional groups

react with permanganate resulting in inactivation they are some-

how blocked when the substrates sites are occupied by carbamyl phosphate and other compounds containing phosphate moiety and also by the aspartate analogue, succinate. The simplest mechanism for such an effect would be that the bound molecules

themselves obstruct the approach of permanganate to the criti- cal functional groups. lNore complex processess involving conformational changes of the protein can also be imagined.

Characterization of Chemical Changes of Catalytic Subunit upon

Treatment with KMn04-The amino acid compositions of catalytic subunit and catalytic subunit which had been inactivated

by treatment with a g-fold molar excess of I<R9n04 are shown in Table III. Inspection of the data revealed that significant changes are seen only in the half-cystine and cysteic acid contents. Apparently, treatment of catalytic subunit with per-

TABLE III

Amino acid composition of catal& subunit and permanganate- treated catalytic subunit

Five milliliters of a 11.1 ELM solution of catalytic subunit in 0.01 M imidazole acetate, pH 7.0, was mixed with 0.05 ml of 6.66 mM KMnOe. The reaction was permitted to proceed for 60 set, at 1%.hich time 0.05 ml of 1 M mercaptoethanol was added. An identical control was mixed with water instead of KMn04. At this point aliquots of both solutions were assayed for enzymatic activity. The specific activity of t,he control was 0.46 moles per min per g and that of the permanganate-treated catalytic subunit was 0.01 mole per min per g. The solutions were exhaustively dialyzed under nitrogen against 0.01 M pyridine. Appropriate aliquots of the dialyzed proteins were taken for acid hydrolysis, alkaline hydrolysis, and half-cystine determination as described under “Materials and Methods” but with omission of the mercaptoethanol from the aminoethylation reaction mixture. The results of the analyses are expressed in number of residues of each kind of amino acid per polypeptide chain of catalytic subunit.

Amino acid

Cysteic acid Methionine sulfoxide.. Aspartic acid. Methionine sulfone. Threonine Serine Glutamic acid Proline Glycine Alanine. Cysteine.......... Valine. Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan Lysine. Histidine. Ammonia. Arginine

I-

Cat&tic subunit KMn04-treated molar ratio catalytic subunit

(per 33,000 g) molar ratio

0 0.84 -0.1 -0.1

33.6 33.8 0 0

15.7 15.7 17.0 17.0 25.4 25.7 10.9 10.8 13.5 13.6 31.1 31.1

0.83 0 18.2 18.6

0.98 7.09 10.96 11.3 35.3 35.2

6.72 6.96 10.6 10.81

2.14 2.11 13.7 13.5

9.86 9.8 34.7 38.0 13.9 13.6


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Reaction of Aspartate TI-anscarbamylase Catalytic Subunit with Pemanganate Vol. 246, No. 10

MOLAR RATIO OF MnO;:CSU TIME (HRi

FIG. 3 (left). Oxidation with KMn04 was performed as described in the legend t,o Fig. 1. Thiol content was determined as described under “Materials and Methods.” The thiol content of catalytic subunit before treatment with permanganate was 2.99 moles/10O,O00 g of protein. The specific activity was 0.530 mole oer min ner g. 0, per cent initial specific activity; a, per cent initial thiol content: CSU, catalytic subunit.

FIG. 4 (riaht). Inactivation of catalytic subunit by sodium tetrathionate. ‘Of an 11.7 mg per ml solution of catalyt,ic subunit in 0.2 M Tris-acetate, 0.001 M disodium EDTA, pH 8.2,2.0 ml were mixed with 2.0 ml of freshly prepared 0.2 M sodium tetrathionate in 0.2 M Tris-acetate, 0.001 M disodium EDTA, pH 8.2. The tube was flushed with nitrogen and t,he tube was incubated at 28”. At suitable times 0.5-ml portions of the react,ion mixture were withdrawn and separately dialyzed at 4” under a, nitrogen atmosphere against 0.2 M Tris-acetate, 0.001 M disodism EDTA, pH 8.2. The dialyzed solutions were assayed for enzymatic activity. The protein concentration of each dialyzed solution was determined by measurement of absorbance at 280 mp. The “zero” time value for specific activity is low because t.he dialysis step does not stop the reaction rapidly. The cysteine contents of the zero and 90.5- hour samples measured as aminoethylcysteine as described under “Mat,erials and Methods” with omission of the mercaptoethanol from the aminoethylation reaction mixture were 1.6 and 0.0, respectively.

mallganate results in the oxidation of all of the half-cystine to cysteic acid. It is noteworthy that tryptophan, methionine, and tyrosine residues are not noticeably modified by permanganate under these experimental conditions. These three amino acids react extremely rapidly with permanganate under these conditions when present as the model compounds nr-acetyl tryptophan amide, N-acetyltyrosine amide and Wacetyl methionine.3 No doubt structural features of catalytic subunit prevent a hydrophilic species such as permanganate from approaching the side chains of these amino acid residues.

All of the half-cystine of catalytic subunit is present as cys- tcinyl residues (16). Thus, the extent of oxidation of the half- cystine of catalytic subunit could be conveniently monitored by the chromogenic reaction of thiols with DTNB originally devised by Ellman (19). By using this reagent as described under “Materials and Methods” it was possible to make accurate comparisons between the extent of oxidation of the protein thiol groups and the extent of inactivation of the enzyme. The results of an experiment designed to make such a comparison are shown in Fig. 3. Identical amounts of catalytic subunit were inactivated to varying extents with various amounts of permanganate and then assayed for activity and thiol content. The curve connects the data points for thiol content as a function of amount of permanganate used for inactivation of catalytic subunit. The open triangles represent the specific activities of the same samples of permanganate-treated catalytic subunit. The close agreement between the two properties of the treated

3 W’. F. Benisek, unpublished observations.

samples would indicate that it is the oxidation of the thiol groups of this protein which is the chemical event responsible for inactivation of the enzyme. This conclusion is reasonable in view of the reported sensitivity of the activity of catalytic subunit to certain very reactive sulfhydryl reagents such as DTNU and p-hydroxymercuribenzoatc (16) as well as tetrathionate ions as shown in Fig. 4.

Oxidation of a thiol group to a sulfonic acid group requires the abstraction of 6 electrons by the oxidizing agent. In order to so oxidize the three thiol groups of catalytic subunit, a total of 18 electrons need to be removed by yermanganate. The extrapolated equivalence point for the inactivation of catalytic subunit by permanganate was 6 permanganate ions per molecule of catalytic subunit of molecular weight 100,000, or 2 per polypeptide chain. Since reduction of a permanganate ion to manganese dioxide consumes 3 electrons, a total of 18 electrons are consumed by the 6 permanganate ions. Thus we can account for all of the permanganate reduction by oxidation of the enzyme thiol groups to sulfonate groups, which further supports the notion that it is oxidation of the thiols which causes inactivation of the enzyme.

Attempted Reversal of Inactivation of Catalytic Subunit by Re- ducing Agents-The inactivation of catalytic subunit by permanganate is not easily reversed; this result is to be expected since sulfonate groups are quite stable to reduction. Permanganate- inactivated catalytic subunit was allowed to incubate at 25” in 0.2 M Tris-acetate buffer, pH 8.2, containing 0.1 M 2-mercaptoethanol for 18 hours. Xssay of the protein after such treatment showed no detectable reappearance of catalytic properties. Similarly, treatment of pcrmanganate-inactivated catalytic subunit with 0.0265 x NaT%H4 in 0.2 M Tris-acetate, pH 9.0, for 30 min at 0” also failed to restore the enzyme’s activity. The lack of reversibility of the oxidation by treatment with mercaptoethanol and borohydride would suggest that no significant amount of the oxidized sulfur atoms was present as sulfinate (20) groups.

Reactivi& of Catalytic Subunit-Thiol Groups toward Permanga- nate-The choice of permanganate as a reagent for site-specific modification of catalytic subunit was suggested by its structural resemblance to phosphate ion. If, indeed, permanganate oxidation of catalytic subunit was achieved via an initial binding of permanganate in a site near the thiol group of each polypeptide chain then one might expect that the thiol groups of the enzyme Tvould be very reactive towards permanganate. The data of Fig. 2 show that the inactivation reaction is a rapid one. It was of interest to determine whether the thiol groups were unusually reactive, typically reactive or hyporeactive and thus some comparison between the reactivity of the protein thiol group and a typical thiol such as 2-mcrcaptoethanol toward permanganate was required. Preliminary experiments soon established that, like catalytic subunit, 2-mercaptoethanol is oxidized by permanganate much too rapidly to measure the rate of the reaction at reactant concentrations high enough to follow the decrease of permanganate spectrophotometrically or the disappearance of thiol by the DTNB reaction. In principle the rate could be decreased by diluting both thiol and permanganate but, unlike catalytic subunit, mercaptoethanol has no catalytic properties which would permit its measurement at the very low concentrations required for slowing the rate sufficiently. Instead, we have sought to determine the ratio of the rates of reaction of permanganate with catalytic subunit on the one hand


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z 5 i= 0 I ::

z 0.2

Y

z5 0.4 2 i= f 0.6

k g 0.8 i=

I I I I 1

MOLE RATIO MERCAPTOETHANOL

TO CSU THIOL GROUPS

FIG. 5. Relative rate of oxidation of catalytic subunit and mercaptoethanol. Four solutions containing 0.117 PM catalytic subunit (0.351 PM catalytic subunit thiol groups) were prepared in 0.01 M imidazole acetate, pH 7.0. They also contained, respectively, 5, 10, and 20 .UM mercaptoethanol. A suitably sized aliquot was removed from each solution and assayed for enzyme activity. These activities were taken as the initial specific activit,ies of the enzyme in each solution. To each solution was then added 0.001 volume of a 0.6 mM solution of potassium permanganate in water. After incubation for 10 min at 28”, aliquots of each solution were withdrawn and assayed in triplicate. The observed specific activities were corrected for the small amount of activity remaining in the mercaptoethanol-free reaction mixture resulting from the fact that slightly less permanganate t,han required for complete inactivation was used. The fraction of the initial specific activity which remained after permanganate treatment was calculated from t,hese values and the corresponding initial specific activities and plotted against the mole ratio of mercaptoethanol to catalytic subunit thiol groups. CSU, catalytic subunit.

and mercaptoethanol on the other. To this end various mixtures of catalytic subunit and mercaptoethanol containing a constant amount of catalytic subunit and variable amounts of mercaptoethanol were treated with an amount of permanganate which was sufficient to inactivate about 90% of the catalytic subunit in the absence of mercaptoethanol. It was reasoned that, with increasing molar ratio of mercaptoethanol to catalytic subunit thiol groups, a greater and greater fraction of the added permanaganate would be diverted to oxidation of mercaptoethanol, thus leaving less and less permanganate available for oxidation of catalytic subunit. The ratio of mercaptoethanol to catalytic subunit thiol in a solution in which half of the catalytic subunit was inactivated when it was treated with an amount of permanganate which would just completely inactivate the catalytic subunit in the absence of mercaptoethanol would equal the ratio of the rates of reaction of catalytic subunit with permanganate and mercaptoethanol with permanganate. For example, if the rates of reaction of catalytic subunit and mercaptoethanol with permanganate were equal then one would expect that treatment of a 1 :l mixture of catalytic subunit and mercaptoethanol with the stoichiometric (with respect to catalytic subunit only) amount of permanganate would at completion of all reaction produce a 50% inactivation of the catalytic subunit. However, if catalytic subunit were 10 times as reactive toward permanganate as mercaptoethanol then one would have

TABLE IV Reactivity of vurious chemical reagenls toward thiol groups of

catalytic subunit

Most of the reagents were tested by Vanaman and Stark (16). Treatment of catalytic subunit n-ith 1 mu Hz02 or 1 mM K,Fe(CN)G for GO min in 0.01 M imidazole acetate, pH 7.0, at 28” was performed. The enzyme was then assayed for specific activity. No difference was detected between the specific activities of the treated samples and a simultaneously treated control lacking reagent. Inactivation of the enzyme by 0.1 M tetrathionate was performed as described in the legend to Fig. 4.

Reagents which do not appreciably react with thiol groups of catalytic subunit

Methyl iodidea

Iodoacetatea Iodoacetamidea Bromomalonatea DL-Bromosuccinatea 2-Bromoethanola Acrylonitrilee N-Ethylmaleimidea Ethyleneiminea 2-Hydroxy-5-nitrobenzyl bro-

midea Carboxymethyldisulfidea Carboxypropyldisulfide” 2.Hydroxyethyldisulfide” 2.Hydroxypropyldisulfidea Hydrogen peroxide” Ferricyanideb

Q Reference 16. h This study.

eagents which reacl less rapidly with catalytic subunit

than with nercaptoethanol

p-Hydroxy- mercuribcn- eoat,ea

DTNBa Tetrathionateb

Reagents which react more rapidly with catalytic subunit

than with mercaptoethanol

to add 10 times as much mercaptoethanol in order to obtain the 50% inactivation. As the reactivity of catalytic subunit ill- creases relative to mercaptoethanol, more and more mercaptoethanol is required to compete equally with the catalytic subunit for the limited amount of permanganate available. The results of such a series of oxidations are summarized in Fig. 5. As expected, increasing the amount of mercaptoethanol exerted a sparing effect on catalytic subunit remaining after permanganate oxidation. Linear interpolation of the data indicated that at a mercaptoethanol to catalytic subunit thiol mole ratio of 45:l the catalytic subunit activity is spared 507,, thus im- plying that the thiol groups of catalytic subunit are 45 times as reactive as the thiol groups of 2-mercaptoethanol.

Hyperreactivity of the protein thiol groups could be due to more than one factor. (a) Permanganate ion functions as a true “affinity reagent, ” interacting specifically with the phosphate binding site which contains or is in close proximity to the thiol group, thus augmenting the rate by a proximity effect. (b) The thiol groups of catalytic subunit are, because of their environment, intrinsically more reactive to sulfhydryl reagents in general. I f the latter explanation were true one should expect that the sulfhydryl groups of catalytic subunit would be readily modified by a wide variety of chemical reagents, but the recent report by Vanaman and Stark (16) convincingly demon- strates that this is not so. These workers tested the ability of a


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315s Reaction of Aspartate Transcarbamylase Catalytic Subunit with Permanganate Vol. 246, No. 10

number of sulfhydryl reagents to react with the thiol groups of catalytic subunit and found that under the conditions which they used, 0.05 to 0.1 M reagent at pH 8.5, the thiol groups either did not react or reacted very slowly relative to the rate of reaction of simple thiols with the same reagents.

These observations of Vanaman and Stark (16), as well as similar results of analogous experiments with additional reagents, are summarized in Table IV. Of the 20 reagents listed in the table, only permanganate is hyperreactive toward catalytic subunit. The reagents which are unreactive or hyporeactive possess a very wide variety of chemical structures and in- clude oxidizing agents, disulfides, and alkylating agents. Thus the thiol groups of catalytic subunit are not intrinsically highly reactive functional groups yet they are readily oxidized by permanganate. Although these data are not conclusive they suggest that permanganate, by virtue of its phosphate-like structure, reacts rapidly because it can bind in phosphate-specific sites near the thiol groups of catalytic subunit.

DISCUSSION

Role of Thiol Groups in Catalysis-The fact that catalytic subunit is inactivated by oxidation of its three thiol groups to sulfonate groups could reflect (a) that these thiol groups are intimate participants in the catalytic event or in substrate binding (or both) or (b) that they are somehow necessary structural elements for maintenance of other functional groups in the cor- rect spatial relationship for their participation in catalysis and binding. Whatever the role is that they play, it is likely that they reside near the carbamyl phosphate binding sites since carbamyl phosphate and, to a lesser extent, other phosphates protect the enzyme against inactivation by permanganate under the mild conditions tested. The recent experiments of Vana- man and Stark (16) make it seem quite unlikely that the thiol groups are essential for catalysis since they have found the S-cyano derivative of catalytic subunit to be fully active. All other chemical modifications of the thiol groups which they prepared (via the mixed disulfide with DTNB) were catalytically inactive. From the limited data available it would appear that introduction of a negative charge near the thiol sulfur or attach- ment of a bulky group to it yields an inactive enzyme while a small neutral group such as --SCN is a tolerated addition. Other chemically modified derivatives of catalytic subunit need to be examined in order to define better the tolerable range of modifications. Hopefully the results of such studies can il- luminate the role that these thiol groups play. Tentatively, one can assume that the role of the thiol groups is indirect, perhaps involving the maintenance of an appropriate active site structure involving other functional groups. It is conceivable that the thiol groups play no important role at all other than to fill an otherwise empty pocket or space near the active site. When negative groups are attached to the sulfur atom as in the S-sulfo derivative (16) or in the sulfonate and sulfcnylthiosulfate derivatives prepared in this work, these negative groups may interact with and block the (presumably) positively charged groups in the active site which recognize the negatively charged substrate molecules. The sheer bulk of the thionitrobenzoate or mercuribenzoate groups might produce a similar occlusion of the active site. Binding studies of substrates and substrate analogues to appropriate chemical derivatives are needed to examine this point. Perhaps mutants of E. coli can be found in

which other amino acid residues replace cyst&e in the catalytic subunits of this enzyme.

The utility of permanganate as a reagent for selective oxidation of one protein’s functional groups has been shown by the experiments reported here. That the action of this reagent is directed toward the active site region is suggested by the pro- tective effect of substrates and substrate analogues and by the fact that the oxidation has a dramatic effect on the activity of the enzyme. Whether permanganate reacts as a true affinity reagent in which reaction is preceded by binding of the reagent in the phosphate binding site is less clear. The hyperreactivity of the protein’s thiol groups toward permanganate in spite of their sluggish reactivity toward other chemical reagents which do not have phosphate-like structures would seem to support such a mechanism. It should be noted, however, that the chemical mechanism of thiol oxidation by permanganate is unknown but presumably involves several intermediate states of oxidation for the thiol sulfur and reduction for the manganese.4 In principle, one should detect prior complex formation of permanganate with the enzyme by a deviation of the order of the reaction in permanganate to values less than 1 as the permanganate concentration is raised. An attempt to do this was frustrated by the great rapidity of the reaction, prohibiting good estimates of the initial velocity by conventional kinetic techniques. But preliminary estimates of the dependence of rate of inactivation of the enzyme on permanganate concentration3 have shown that the rate increases only 80% when the permanganate concentration is increased by 300%.

The applicability of permanganate to probing phosphate binding sites in other proteins is at present unknown. The wide occurrence of such binding sites in enzymes should provide ample material for assessing this aspect. Other tetrahedral oxyanions such as chromate may prove to be useful reactive phosphate analogues. Dichromate ion is a close structural analogue of pyrophosphate, another anion often recognized by enzymes. Percarbonate, eOzC-O-O-CO~e, might mimic dicarboxylic acids such as succinate, aspartate, and so on. Perusal of modern texts in inorganic chemistry provides other structural analogies.

Aclcnowledgments-The author wishes to express his thanks to Dr. George Stark for many hours of stimulating discussion and to Dr. Thomas Vanaman and Kim Collins for numerous

helpful suggestions and discussions. The careful technical as- sistance of ;VIrs. Sija Kim is gratefully acknowledged.

REFERENCES

1. GERHART, J. C., AI\‘D PARDEE, A. B., J. Biol. Chem., 237, 891 (1962).

2. GERHART, J. C., AND PARDEE, A. B., Fed. Proc., 23, 727 (1964). 3. PORTER, R.W., MODEBE, M. O., AND STARK, G. R., J. Biol.

Chem., 244, 1846 (1969). 4. SCHMIDT, P. G., STARK, G. R., AND BALDESCHWIELER, J. D.:

J. Biol. Chem., 244, 1860 (1969).

4 Species of manganese at lower states of oxidation than permanganate such as manganate (MnOh-) and hypomanganate (MnOP) may be intermediates in the reduction of MnO, by catalytic subunit, but these ions are extremely unstable in neutral solution, disproportionating rapidly to permanganate and manganese dioxide. It is possible, in view of the phosphate-like structure and charge of these ions, that they might act as oxidizing agents which could bind in the phosphate binding site of the enzyme and oxidize functional groups in or near the site.


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chemistry, Acadeinic Press, New York, 1965, p. 1. 20. MOORE, S., AND STEIN, W. H., in S. P. COLOWICK AND N. 0.

KAPLAN (Editors), Methods in enzymology, Vol. VI, Aca- demic Press, New York, 1963, p. 819.

COLE, R. D., in C. HIRS (Editor), Methods in enzymology, Vol. XI, Academic Press, New York, 1967, p. 315.

VANAMAN, T. C., AND STARI<, G. R., J. Biol. Chem., 245, 3565 (1970).

SEDLAK, J., AND LINDSAY, R. H., Anal. Biochem., 25, 192 (1968).

HAMMIB, G. G., PORTER, R. W., AND Wu, C.: Biochemistry, 9, 2992 (1970).

ELLMAN, G. L., Arch. Biochem. Biophys., 74, 443 (1958). TRUNDLE, D., AND CUNNINGHAM, L. W., Biochemistry,8,1919

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William F. Benisekwith Permanganate Ion, a Reactive Structural Analogue of Phosphate Ion

Aspartate TranscarbamylaseEscherichia coliReaction of the Catalytic Subunit of

1971, 246:3151-3159.J. Biol. Chem.

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