THE JOURNAL OF BIOLOQICAL CHEMISTRY Vol. 249, No. 9, Issue of May 10, pp. 2343-2351, 1974

Printed in U.S.A.

Modification of Essential Histidyl Residues of the ,& Subunit of

Tryptophan Synthetase by Photo-oxidation in the Presence of

Pyridoxal 5’-Phosphate and L-Serine and by Diethylpyrocarbonate*

(Received for publication, August 22, 1973)

EDITH WILSON R~ILES~ AND HIDEHIKO KUMAGAI$

From the Laboratory of Biochemical Pharmacology, National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, Bethesda, Maryland SO014

SUM MARY

Photoinactivation of the fiZ subunit of tryptophan synthe- tase is dependent on the presence of the coenzyme, pyridoxal S/-phosphate, and results in the specific destruction of 1 his- tidy1 residue per /3 monomer. The rate of photoinactivation is strikingly increased by the addition of a substrate, L-serine. Inactivation of the pz subunit can also be achieved by the modification of 2 to 3 histidyl residues per monomer with diethylpyrocarbonate. pZ subunit modified by either pro- cedure can still form a complex with pyridoxal 5’-phosphate. The chemically modified 02 subunit can form some Schiff base intermediate between pyridoxal St-phosphate and L-serine but appears to be blocked in the further conversion of this intermediate. These results suggest that 1 of the chemically modified histidyl residues may serve a role in the abstraction of the a-proton of L-serine. This suggestion is supported by the finding that the Vmax for the conversion of L-serine to pyruvate is dependent on an ionizing group with a pK of 6.7.

Tryptophan synthetase of Escherichia coli is a multienzyme complex composed of two proteins which were originally termed the A and B components (2). These readily separable proteins are now termed the LY and f12 subunits, respectively, since the CY subunit exists as a single chain in solution and the /?z subunit normally exists as a dimer in solution. These subunits combine to form an o(& co~nples which carries out Reactions 1 to 3 (3). The pZ subunit, which contains 2 moles of pyridosal-P, catalyzes Reaction 4 (4), and a number of other l)yridosal-I’-depciidclit /3 elimination and fi replacement reactioiis (5).

* A preliminary report of some of this work was presented at the 57fh Annual-Meeting of the American Society of Biological Chemists. Atlantic Citv. New Jersev. Anril 1973 (1).

1 To whom reprint requests shoulh’be-addressed.’ 0 Present address, The Research Institute for Food Science,

Kyoto University, Uji, Kyoto, Japan.

Indole + L-serine * L-tryptophan

Indole&glycerol phosphate e

(1)

indole + n-glyceraldehyde 3-phosphate (2)

Indole%glycerol phosphate + L-serine +

L-tryptophan + n-glyceraldehyde 3-phosphate (3)

L-Serine ---) pyruvate + ammonia (4)

The pz subunit alone has a small activity in Reaction 1 which is greatly stimulated by the presence of a high concentration of ammonium ions (6) or by addition of the LY subunit. Since the cx subunit alone has a small activity in Reaction 2, it is thought that the LY subunit provides the catalytic site for the hydrolysis of indole-3-glycerol phosphate to indole and D-&C-

eraldehgde S-phosphate in both Reactions 2 and 3, and that the flZ subunit further converts the indole to tryptophan iu Reaction 3 (3).

Recent studies iu our laboratory (1, 7) have shown that the conversion of L-serine to pyruvate by the PZ subunit iu the ab- sence of NH4+ ion is rate-limited by the removal of the cu-proton of L-serine; a fluorescent enzyme-substrate intermediate accu- mulates before this rate-limiting step. Lysiue or histidine residues may have catalytic roles in the removal of the cu-proton in reactions catalyzed by pyridosal-1’ enzymes (8). This paper therefore looks for kinetic and chemical evideuce for a possible catalytic role of a histidiuc residue in reactions of the & subunit. Crawford and co-workers have recently reported the presence of 2 histidyl residues iu the “active site peptidc” of the pZ sub- unit of tryptophan syuthetasc isolated from E. coli and Pseu- domonas pulida (9, 10).

We have investigated the photoiuactivation of the & subunit in the presence of pyridosal-P since it has been previously found that pyridoxal-I’ can act as a photoseusitizer for a single histidyl residue per polypeptide chaiu of 6-phosphogluconate dehydro- genase aud rabbit muscle and spinach aldolases at ucutral pH (11-13). These investigators demonstrated that binding of pyridoxal-1’ to a specific site on each of these proteins was neccs- sary for the observed photoiuactivation (11-13). Scoffone aud co-workers have proposed and presented some evidence that a natural chromophore bound to a protein or a covalcntly bound

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a rl

H H c=c-R

of a high concentration of hydroxylamine (25) for enzyme from

CH,CH,OC-0-C-OCH+H, + / \ -

photoinactivation experiments. Hydroxylamine has been found to greatly increase the activity of the 01& complex in Reaction 2

‘NC/NH (27). However, this assay cannot be used for diethylpyrocar- bonate-modified @Z subunit, since hydroxylamine reverses the

H modification. (26).

Reaction 3 was assayed spectrophotometrically The serine deaminase activity of the 6~ subunit (Reaction

Diethylpyrocarbonate Histidyl 4) was assayed spectrophotometrically; pyruvate formation was coupled with lactic dehydrogenase and DPNH (4).

Treatment with Diethylpyrocarbonate-Diethylpyrocarbonate was freshly diluted to 0.01 M with 690 volumes of cold ethanol for each experiment.

H H Protein solutions (about 1 mg per ml) in 0.1 M

CH CH 0-i-i

--C-R potassium phosphate buffer, pH 6.5, were treated with diethyl-

’

pyrocarbonate in a Cary 14 spectrophotometer cooled with circu-

CH,CH,OH + CO2 + lating water at 4”. The modification of histidyl residues was fol-

3 2 \c//”

lowed by measuring the difference in absorbance at 242 nm be- tween the treated sample in the sample cell and a control enzyme

H solution in the reference cell which was treated with an equal

Carbethoxyhistidyl (242 nm) volume of cold ethanol (usually 2.5yo final concentration). The number of modified histidyl residues was calculated using a value

SCHEME 1 of 3,200 M-l cm-i for the extinction coefficient at 242 nm of N-car- bethoxyhistidyl residues in proteins (19), assuming that this value

sensitizing dye may result in the selective modification of the applied to modified histidyl residues of the o2 subunit.

potentially photo-oxidizable amino acid residues which are Reversal of Diethylpyrocarbonate Modification with Hydroxyla-

mine-Aliquots of reaction mixtures with diethylpyrocarbonate

adjacent to the sensitizer within the molecule (14-15). We and untreated controls (0.1 ml) were incubated with 0.3 ml of 0.1

have also tried chemical modification by diethylpyrocarbonate M potassium phosphate, pH 7.0, containing 1 M NH%OH.HCI

which acylates a histidyl residue by the reaction shown in Scheme (adjusted to pH 7.0 with KOH), 10 mM p-mercaptoethanol, and 1

1; treatment with hydroxylamine regenerates the free histidyl mM EDTA for 20 hours at 22’; the solutions were dialyzed for 6

residue (16, 17). hours against four changes of 0.1 M potassium phosphate buffer,

Several enzymes have been reversibly modified pH 7.8, containing 10 mM @-mercaptoethanol and 1 mM EDTA

by this reagent (18-21). before assay. One solution was incubated and dialyzed similarly except that NHtOH was omitted (untreated control).

EXPERIMEXTAL PROCEDURE Spectra-Absorption spectra were recorded in a Cary model 11

or Cary model 14 recording spectrophotometer. Circular dichro-

Materials ism and optical rotatory dispersion measurements were made in

Pyridoxal-P, 5,5’-dithiobis(2.nitrobenzoic acid), and DPNH a Cary model 60 equipped with a circular dichroism attachment

were purchased from Sigma Chemical Co. Guanidine hydro- (model 6001) in 3-ml cells with a 1.0.cm path length at 27”. Mean

chloride and urea (ultrapure) were products of Schwarz-Mann. residue ellipticity values [e] mrw were calculated by using the ex-

Lactic dehydrogenase was a product of Worthington Biochemical pression [B],,, = (S)m/lc’. 10, where m is the mean residue molecu-

Corp. Diethylpyrocarbonate (diethyloxydiformate) was ob- lar weight of the sample, 0 is the observed ellipticity, 1 is the path

tained from Eastman Organic Chemicals. length of the sample solution in centimeters, and c’ is the concen- tration in grams per cm3. [elm,, has the dimensions of deg cm2

Methods per dmole of amino acid residue. The mean residue molecular weight for the 82 subunit was calculated to be 108 from the amino

Enzyme Preparations and Assays-The pz subunit of tryptophan acid analysis data of Goldberg et al. (28). Fluorescence measure- synthetase was purified from the A2/F’A2 strain of E. coli by ments were made in an Aminco-Bowman spectrofluorimeter on either the standard method of Wilson and Crawford (Preparation O.l-ml volumes of solutions in a rectangular microcell with a 3-mm I) (2) or by the new method of Adachi andMiles (Preparation II).] cross-section. APO+& subunit was prepared as described previously (22). The Determination of Enzyme-bound Pyridoxal-P--B, subunit which molecular weight of the @ monomer used for calculations was 44,500 (23).

had been dialyzed against 0.1 M potassium phosphate, pH 7.8, The a subunit was prepared by a modification (6) of containing 2 X 10e6 M pyridoxal-P, was treated with 4 volumes of

the standard method (24). All assays were performed at 37” un- 10% trichloroacetic acid and a 1’ less otherwise stated. A unit of activity in any reaction is the reagent of Wada and Snell (29).

,zo volume of the phenylhydrazine

disappearance of 0.1 rmole of substrate or the appearance of 0.1 The mixture was centrifuged and

the absorbance of the supernatant solution at 412 nm was read rmole of product in 20 min.

The activities of the p2 subunit and of the CY& complex in Reac- after 10 min against a blank in which enzyme was replaced by an equal volume of the dialysis buffer. The concentration of bound

tion 1 were measured as described previously (7, 25). Reaction 2 pyridoxal-P was calculated from the molar extinction coefficient was assayed by the spectrophotometric assay (26) for enzyme (edI nm = 24,700 M-I cm-l) (29).

modified by diethylpyrocarbonate or by the assay in the presence Photoinactivation-The photoinactivation experiments were

i We wish to thank Mr. David L. Rogerson for growing large carried out at 25” with the enzyme solution (1 to 3 mg per ml) in

batches of cells and Dr. Osao Adachi for preparing the pZ subunit either a 13 X loo-mm tube (for sample volumes of 1 ml) or a 15 X

used for the photoinactivation experiments. The new method 150-mm tube (for sample volumes of 2 ml). The Pyrex tube was

of 0. Adachi and E. W. Miles results in homogeneous, crystalline placed 7 cm from a 500-watt Viewlex projector. A flat sided Pyrex

02 subunit without steps in which the enzyme is heated (Prepara- bottle filled with water was placed between the sample tube and

tion II). Details of the method are available as JBC Document the projector and took up about 6 cm of the light path. This

No. 73M-1217, in the form of one microfiche or 4 pages of phot’o- bottle served to shield the sample from any heat or ultraviolet light coming from the lamp.

copy. Orders for supplementary material should specify the title, The sample was stirred with a small

authors, and reference to this paper and the JBC Document num- magnetic stirring bar. In some experiments aliquots were re-

ber, the form desired (microfiche or photocopy), and the number of moved at various times for determination of enzymatic activity.

copies desired. Orders should be addressed to the Journal of Bio- In other experiments each sample was illuminated for the stated

logical Chemistry, 9650 Rockville Pike, Bethesda, Maryland 20014, time and then removed for further analysis.

and must be accompanied by remittance to the order of the Journal Amino Acid Analyses-Samples for amino acid analysis were

in the amount of $2.50 per microfiche or per photocopy. A much precipitated with 10% trichloroacetic acid, washed three times

more rapid method has subsequently been developed (0. Adachi each with 5’% trichloroacetic acid, acetone, and ether, and dried

and E. W. Miles, manuscript in preparation). under a stream of nitrogen. Hydrolyses were carried out in sealed, evacuated tubes with 5.7 N HCl for 24 hours at 110” and

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were then analyzed on a Beckman Spinco model 120C amino acid analyzer using the standard accelerated (120 ml per hour) sys- tem (30).2

Tryptophan was determined by the spectrophotometric method of Edelhoch (31) in samples precipitated as above; the protein (about 2 mg) was redissolved in 1 ml of 6 M guanidine HCl in 0.02 M potassium phosphate, pH 6.5. Since the protein recovery was not quantitative, the number of tyrosyl residues per monomer was assumed to be 11 and the number of tryptophyl residues and the protein concentration were calculated -from two simultaneous eauations (31). Methionine sulfoxide was determined after alka- line hydrolysis (32). Samples of protein (1 to 2 mg) were lyophil- ized from 0.01 M NHdHC03 in polypropylene tubes (10.7 X 75 mm) which were used as liners inside vacuum hydrolysis tubes (12 mm (inside diameter) X 120 mm) (Kontes) (33) ; 0.5 ml of 3.75 N NaOH was added and tubes were evacuated and heated for 16 hours at 110”. The polypropylene liners were used to prevent the solu- bilization of silica from the glass tubes by alkali (33). The pH was adjusted to 1.5 with HCl before analysis. Histidine and tyro- sine were determined with diazonium-lH-tetrazole on protein samples which were first denatured in 0.5 N NaOH (34). This method permits the determination of the concentrations of bisazo- histidine and bisazotyrosine in the same solution from absorbance values at 480 nm and 550 nm using a set of simultaneous equations (34).

RESULTS

Photoinactivalion of the pZ Subunit-Illumination of solutions of the 02 subunit in the presence of pyridoxal-P results in loss of activity which follows pseudo-first order kinetics (Fig. IA, open

symbols). Addition of L-serine causes a 4-fold increase in the rate of inactivation (Fig. lA, closed symbols, and Fig. 1B). The rates of photoinactivation in the presence and absence of L-

serine were pa-dependent and the data points in plots of the pseudo-first order rate constants versus pH fall close to theoretical titration curves calculated for pK values of 6.9 (Fig. 1B). Illu- mination of the apo-Pz subunit either in the presence or absence of L-serine had little effect on the activity assayed after recon- stitution with pyridoxal-P.

The pH dependence of the photoinactivation is suggestive evidence that a histidyl residue is being photo-oxidized since the dye-sensitized photo-oxidation of histidine has been shown to be sharbly pH-dependent, the protonated form of histidine being resistant to oxidation (35). However, the observed pH effect could also be due to the ionization of some group on the pyri- doxa,l-P Schiff base (12).

Fig. 2A shows the effect of the time of illumination of the & subunit in the presence of L-serine and pyridoxal-I’ on its ac- tivity, total sulfhydryl content, and absorbance at 425 nm. No change in total sulfhydryl residues which could be titrated with 5,5’-dithiobis(2-nitrobenzoic acid) in guanidine hydrochloride was observed. The observation that the absorbance of the enzyme solution at 425 nm decreased during illumination indi- cates that some photodestruction of enzyme-bound pyridoxal-1’ occurred. However, the rate of this destruction (k’ = 0.089 min-I) was slower than the rate of inactivation (k’ = 0.23 min+).

Fig. 2B shows some properties of the enzyme solutions from Fig. 2A after overnight dialysis against 0.1 M potassium phos- phate buffer containing 0.02 mM pyridoxal-1’. Although this dialysis had no effect on the activities of the illuminated enzyme solutions (not shown), it did result in the displacement by fresh pyridoxal-P of the pyridoxal-1’ which had undergone photode- struction.

Absorbance spectra of the samples after dialysis were closely similar (not shown). However, the samples which had been il-

2 These analyses were kindly performed by Mr. George Poy, Arthritis and Rheumatism Branch, National Institute of Arthritis, Metabolism, and Digestive Diseases.

5 IO 15 20 25 30 MINUTES

k’ 0.2

0. I

DH

FIG. 1. Effect of pH and L-serine on the rates of photoinactiva- tion of the 0~ subunit in the presence and absence of pyridoxal-P. A, activity loss of holoenzyme in the presence of L-serine at pH 8.3 (o), pH 7.1 (A), pH 6.6 (m); activity loss of holoenzyme alone at pH 8.8 (0) or pH 6.96 (A) ; and activity loss of the apoensyme at pH 7.8 in the presence of L-serine (O ) and in the absence of L-serine (A). B, effect of pH on the pseudo-first order rate con- stant for photoinactivation in the presence (0) and absence (A) of L-serine. Each rate constant was calculated from a pseudo- first order plot of the activity loss such as one of those shown in A. The solid lines are theoretical titration curves calculated for a pK of 6.9. Preparation II (see “Experimental Procedure”) of the pZ subunit was dialyzed at 20 mg per ml against 0.1 M potassium phosphate, pH 7.8, containing 1 mM EDTA, 0.02 M pyridoxal-P, and 0.01 M p-mercaptoethanol. Aliquots (0.05 ml) were diluted to 0.85 ml with 0.1 M potassium phosphate buffers of various pH values, each containing 0.025 mM pyridoxal-P and 1 mM EDTA. Water (0.1 ml) or L-serine (0.1 ml of 1.0 M) was added just before illumination to bring the final volume to 1 ml and the protein concentration to 1 mg per ml. Aliquots (0.02 ml) were removed at the indicated intervals after the light was turned on and added to the reaction mixture for the assay of serine deaminase activity. After each sample had been illuminated, the pH was determined. APO-& subunit was dialyzed against 0.1 M potassium phosphate, pH 7.8, containing 1 mM EDTA before illumination in the presence and absence of 0.1 M L-serine at a final protein concentration of lmlperml.

luminated for increasing times showed a small progressive shift to longer wavelengths in their spectra in the visible range. The absorption maximum of the control was 412 nm while that of enzyme illuminated for 13 min was 420 nm. Fig. 2B also shows the fluorescence properties of these samples which had been il- luminated and then dialyzed. The fluorescence emission at 500 nm of each sample was measured before (A) and after (0) the addition of L-serine. Untreated /3z subunit has a low fluores- cence in the absence of L-serine which increases IO-fold on the addition of L-serine to a value which is arbitrarily called loo’%. Illumination causes a progressive increase in the fluorescence of the pyridoxal-P bound after dialysis. Illumination also causes a progressive decrease in the fluorescence in the presence of L-

serine.

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60

60

--I

--1

0 2 4 6 6 IO 12 14 MINUTES

FIG. 2. Effect of the time of illumination on various properties of the fit subunit. Preparation II of the 02 subunit was dialyzed against 0.1 M potassium phosphate, pH 7.8, containing 0.02 mM

pyridoxal-P and 1 mM EDTA, and then diluted with this buffer containing 0.1 M L-serine to give a final protein concentration of 3 mg per ml and a final volume of 1 ml. Each sample was illumi- nated for the indicated time immediately after dilution. Each sample was stirred after the light was turned off until a total stir- ring time of 13 min had elapsed. A, serine deaminase activity (0) was determined on 0.005-ml or O.Ol-ml aliquots as described under “Experimental Procedure.” The activity of the control was 1513 units per mg. The absorbance at 425 nm (A) was determined on each sample after illumination against a reference cuvette con- taining the dialysis buffer. The absorbance of the control was 0.41. Total sulfhydryl (SH) content (0) was determined on a O.l-ml aliquot of each sample after dilution with 0.4 ml of 9 M

guanidine HCl, pH 7.8, and the addition of 0.01 ml of 0.01 M 5,5’- dithiobis(2.nitrobenzoic acid) by the method of Ellman (3(i). The sulfhydryl content of the control was 3.53 moles per mole of monomer. Results are expressed as per cent of the control which was not illuminated. B, samples were dialyzed overnight against two changes of 0.1 M potassium phosphate, pH 7.8, containing 1 mM EDTA and 0.02 mM pyridoxal-P. The fluorescence emission of each sample (excite 420 nm, emit 500 nm) was determined before (A) and after (0) the addition of L-serine to 0.1 M. Pyridoxal-P binding (IX) was determined as described under “Experimental Procedure.” Pyridoxal-P, 0.92 moles, was bound per monomer of the control. Pyridoxal-P binding is expressed as the per cent of the control which was not illuminated. The fluorescence of this control in the presence of L-serine is arbitrarily set at 100% and other fluorescence values are expressed as a per cent of that value.

Ej’ect of Photoinactivation of the & Subunit on Its Amino Acid

Composition-Histidine is the only amino acid susceptible to photo-oxidation which was changed during photoinactivation of the & subunit (Table I). Approximately 1 residue of histidine per monomer was found to disappear by reaction with diazonium- 11.I-tetraaole which has been used in the quantitation of both his- tidine and tyrosine residues (34). The recoveries of both tyrosine and histidine in untreated & subunit by the diazonium- II-I-tetrazole method were both lower by 1 to 2 residues than values in the literature obtained from amino acid analysis (37). This is probably due to the incomplete formation of the bisazo derivatives as has been found with glyceraldehyde 3-phosphate dehydrogenase (38).

The rate of decrease of fluorescence in the presence of L-serine No loss of tryptophan was detected as the result of illumina- (k’ = 0.04 min-1) was approximately equal to the rate of loss tion. No change in methionine content was detected after acid of bound pyridoxal-1’ after dialysis. The activities of the photo- hydrolysis, although the amount of this amino acid was consid- inactivated samples and control were also assayed in Reaction 2, erably lower than the literature values (28, 37). The oxidation the conversion of indole-3-glycerol phosphate to indole and of methionine to methionine sulfoxide cannot be detected by glyceraldehyde 3-phosphate in the presence of excess o( protein amino acid analysis of acid hydrolysates since methionine sulfox- (see “Experimental Procedure”). The observed rate of decrease ide is reconverted to methionine under these conditions. Methi- in this activity was approximately the same as the rate of de- onine sulfoxide in proteins can also be reconverted to methionine crease in pyridoxal-P binding and the rate of decrease in the with @mercaptoethanol (32). Native and illuminated PZ sub- fluorescence in the presence of L-serine. The specific activity unit solutions were dialyzed for 24 hours against 0.1 M potassium of the control (814 units per mg) decreased by 29% to 580 units phosphate buffer, pH 7.8, containing 0.02 mM pyridoxal-P and

TABLE I Amino acid analyses of the 0~ subunit before and after photoinactiva-

tion in the presence of pyridoxal-P and L-serine

Amino acid Native p

Literature value

Illuminated p

Ref. 28 Ref. 37

residues/43,000 g

Histidineb. . . . 11.9 (0.1) 11.1 (0.1) 12 14 Tyrosineb.. _. _. . . 9.8 (0.2) 9.7 (0.1) 11.5 11 Tryptophan”. . 1.8 1.9 1 2 Methionined. . . . . . . 10.2 (0.24) 10.4 (0.3) 13 13 Methionine sulfoxidee.. . 0 0 Cysteinef.. . . . . . . . 3.5 3.5 5 5

0 One-milliliter aliquots of the 02 subunit (Preparation II at 2 mg per ml in 0.1 M potassium phosphate, pH 7.8, containing 0.02 mM pyridoxal-P and 0.1 M L-serine) were illuminated for 10 min. The residual serine deaminase activity was 6yo of that of the con- trol.

b Determined with diazonium-lH-teLraeole on proteins de- natured in 0.5 N NaOH (34). A total of 17 analyses on each sample were averaged. Variation is expressed in parentheses as the standard deviation of the mean.

c Single spectrophotometric determination in 6 M guanidine HCl (31). See “Experimental Procedure.”

d The value after hydrolysis in 5.7 N HCl was normalized to leucine = 40 (37). Three analyses were averaged and the stand- ard deviation of the mean is shown in parentheses.

e Single determination after alkaline hydrolysis (see “Experi- mental Procedure”) of samples which had been treated with fi-mercaptoethanol (see “Results”).

f Determined with 5,5’-dithiobis(2-nitrobensoic acid) in the presence of 7 M guanidine IICl as described in Fig. 2.

per mg after 13 min of illumination. These results indicate that three processes occur during illumination: (a) a rapid loss of serine deaminase activity; (b) a slower photodestruction of enzyme-bound pyridoxal-1’; and (c) a still slower process which destroys the ability of the enzyme to bind pyridosal-1’ and to form a complex with the a: subunit which is active in Reaction 2.

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FIG. 3. Effects of diethylpyrocarbonate on the spectrum (A) and on the enzymatic activities and the histidine content of the apo& subunit (B). Apo& subunit (Preparation I at 1 mg per ml in 0.1 M potassium phosphate, pH 6.5) was treated with 0.05 mM or 0.25 mM diethylpyrocarbonate in a Cary spectrophotometer at 4” as described under “Experimental Procedure.” Difference spectra were recorded at intervals between untreated ~2 subunit in the reference cell and treated BZ subunit in the sample cell; an aliquot (0.01 ml) of the treated enzyme was removed immediately after the spectrum was recorded and added to the reaction mixture for assays of serine deaminase activity. A, difference spectra with 0.25 mM diethylpyrocarbonate recorded at 5, 10, 15, 20, 30, 40, and 60 min (-) ; difference spectra with 0.05 mM diethylpyro- carbonate recorded at 3, 5, 10, 20, 30, and 45 min (- - -). The times refer to the time when the spectrum was completed and an aliquot was removed for assay. Spectra were also recorded of the enzyme solutions before (---) and after (------) treat- ment for 60 min with 0.25 mM diethylpyrocarbonate using a buffer blank. The absolute absorbance at 242 nm of the two enzyme solutions was 0.65 and 1.05, respectively. Spectra and difference spectra could not be recorded below 237 nm because the absorbance became greater than 2. B, effect of modification of histidyl resi- dues and subsequent treatment with hydroxylamine on the serine deaminase activity of the 62 subunit. APO-& subunit was treated with0.05 mM (O), 0.1 mM (O), or 0.25 mM (0) diethylpyrocar- bonate as above for various times before determination of the ac- tivity and the.number of histidyl residues modified from the differ- ence absorbance at 242 nm (Curve 1). Aliquots of enzyme treated with 0.1 mM diethylpyrocarbonate were removed at various times, treated with hydroxylamine, dialyzed, and assayed (A) (Curve 2) (see “Experimental Procedure”). The extent of modi- fication before hydroxylamine treatment is shown on the abscissa.

1 M @-mercaptoethanol at 24’ and were then dialyzed against 0.01 M NH4HC03 containing 0.02 mM pyridoxal-P for 24 hours at 4”. The serine deaminase activity of the illuminated fiZ sub- unit increased from 8% of the control to 16% of the control after

treatment of both solutions with B-mercaptoethanol. (The specific activity of the control decreased by 30% under these stringent conditions.) These data, together with the finding of no methionine sulfoxide after alkaline hydrolysis of the B-mer- captoethanol-treated samples (Table I), indicate that only a small part, if any, of the loss of activity on illumination can be attributed to oxidation of methionyl residues. No change in total sulfhydryl residues was detected after illumination, al- though the recovery of total sulfhydryl residues for both samples was low. One to two sulfhydryl residues appear to be readily oxidized when the BZ subunit is stored in dilute solution; this process is accelerated by stirring.

Inactivation by Diethylpyrocarbonate--The spectra of the apo-flz subunit before and after treatment with 0.25 mM diethylpyro-

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tween treated and untreated enzyme solutions during the course of reactions with two different concentrations of diethylpyrocar- bonate are also shown. These difference spectra show absorb- ance peaks at 240 nm which are characteristic of N-carbethoxy- histidyl residues in proteins (see Scheme 1 and Ref. 39). The absence of changes in the difference spectra at 270 nm indicates that no modification of tyrosyl residues had occurred; O-car- bethoxytyrosine absorbs between 270 and 280 nm (16). Fig. 3B shows the results of an experiment in which both the serinc de- aminase activity and the extent of modification of the BZ subunit were followed during treatment with three different concentra- tions of diethylpyrocarbonate. Curve 1 shows that the serine deaminase activity decreased as increasing numbers of histidyl residues were modified. The extrapolated line indicates that loss of activity is associated with modification of 2 to 3 histidyl residues per monomer. Five to six residues were modified at higher concentrations of reagent. Curve d shows the activities of enzyme solutions first modified to the extent shown on the abscissa and then treated with hydroxylamine as described under “Experimental Procedure.” The results show that inactivation was largely reversible and thus due mainly to the modification of histidyl residues. Some irreversible inactivation, particularly at higher concentrations of reagent, must be due to the irreversi- ble modification of other groups, such as lysyl residues. Maxi- mum reactivation by hydroxylamine occurred after 4 hours of treatment. Half-maximal reactivation occurred after 30 to 60 min of treatment. Controls treated with hydrosylamine lost 8 y. activity after 2 hours of treatment and 16% activity after 20 hours.

The modified BZ subunit was quite stable at pH 8.0 for several days. Modified enzyme (28% residual activity) and control enzyme solutions at 1 mg per ml were dialyzed against 0.1 M

potassium phosphate, pH 7.8, or 0.1 M NaHCOs, pH 8.3, both containing 0.1 mM pyridoxal-P, 1 mM EDTA, and 1 mM j3-mer- captoethanol for 96 hours and showed no change in activity. The modified enzyme could still be reactivated to 88% activity by treatment with hydroxylamine after dialysis for 96 hours.

Fig. 4 shows the effect of pyridoxal-P on the rate and extent of modification of histidyl residues. One histidyl residue was pro- tected by pyridoxal-1’ from modification (see difference Curve I-2). Pyridoxal-1’ also decreased the rate of inactivation but did not prevent inactivation.

BJects of Moclijication by Dieth$p?/rocarbonate on the Sulfhydryl Content and Optical Rotation of the /3~ Subunit-Total sulfhydryl content of enzyme solution was determined in the presence of 8 M urea in 0.1 M potassium phosphate, pH 7.8, by the method of Ellman (36). Untreated apo-02 subunit and apo-Bz subunit which had been treated with 1 mM diet.hy1pyrocarbonat.e and had 3% residual activity each contained 4.1 sulfhydryl residues per mole of monomer. These results show that no sulfhydryl residue was modified even though the apo-j3 subunit contains 2 reactive sulfhydryl residues (25). Optical rotatory dispersion spectra of the Bz subunit and many other proteins have a trough with a minimum at 233 nm. Changes in rotation at this wavelength would indicate that changes in LY helical content or in ordered structure have occurred. Untreated apo-Bz subunit had r4,“:, = -5300 f 100. Apo& subunit treated with 0.1 mM di- ethylpyrocarbonate and having 18% residual activity had

bl;:, = -5500 f 100. Therefore, modification did not cause any conformational change which could be detected by this measurement.

Binding of Pyridoxal-P to Modijied BZ Subunit-Fig. 5 shows carbonate are shown in Fig. 3A. Difference spectra made be- absorption spectra (lop) and circular dichroism spectra (bottom)

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MINUTES

FIG. 4. Effects of pyridoxal-P on the rates and extents of inac- tivation and histidyl modification of the 82 subunit. APO-& sub- unit (Preparation I at 0.915 mg per ml in 0.1 M potassium phos- phate, pH 6.5) was treated with 0.25 mM diethylpyrocarbonate as described in Fig. 3. The serine deaminase activity (A) and the extent of histidyl modification (A) (Curue 1) were determined at various times as described under “Experimental Procedure.” APO-& subunit was preincubated with 0.1 mM pyridoxal-P for 1 hour before identical treatment was carried out. Activity, l ; histidyl modification, 0 (Curve 2). Curve i-2 (0) is the difference between the extent of histidyl modification in the absence of py- ridoxal-P (Curve 1) and the extent of histidyl modification (Curve 2) in the presence of pyridoxal-P. PLP, pyridoxal-P.

of untreated and diethylpyrocarbonate-modified enzyme solu- tions. The absorption spectra of the two enzyme solutions be- tween 300 and 500 nm are similar but suggest that slightly less pyridoxal-P is bound after modification. A direct measurement of bound pyridoxal-P (see “Experimental Procedure”) showed that the modified enzyme bound 80% as much pyridoxal-P as the control.

We have previously shown (40) that the circular dichroism spectrum of the pZ subunit shows ellipticity bands between 300 and 500 nm which correspond closely in shape to the absorbance spectrum in this region (see Fig. 5) and to positive ellipticity bands between 260 and 300 nm which are mainly due to aromatic residues. The circular dichroism spectrum of the modified en- zyme between 300 and 500 nm is qualitatively similar to that of the control and indicates that the asymmetric environment of the bound pyridoxal-P is unchanged by modification. The re- duced amplitude of the bands of the modified enzyme is mainly due to a decrease in total bound pyridoxal-P (see above). The control and modified enzymes have closely similar circular di- chroic spectra between 250 nm and 300 nm. This region of the spectrum is mainly due to the aromatic residues of the protein, although the bound pyridoxal-P may also contribute. The data indicate that the asymmetric environment of the aromatic resi- dues is not changed by modification.

Reactions of Modified 02 Subunit with Substrates and with (Y Subunit-The activities of untreated and of modified & subunit were determined in Reaction 1 in the presence or absence of the (Y subunit, in Reactions 2 and 3 in the presence of a! subunit, and in Reaction 4 as described under “Experimental Procedure.”

3000 I I

350 400 450 500

WAVELENGTH,nm

I I I I I

I( 0 a I I I I

300 350 400 450 500 WAYELENGT”.“rn

FIG. 5. Absorption spectra and circular dichroism spectra of untreated and diethylpyrocarbonate-treated 82 subunit in the presence of pyridoxal-P. Diethylpyrocarbonate-treated ape-& subunit (Preparation I containing 2.9 moles of modified histidyl residues per mole of B monomer) and untreated control enzyme were dialyzed against 0.1 M potassium phosphate buffer, pH 7.8, containing 0.1 mM pyridoxal-P, 1 mM &mercaptoethanol, and 1 mM EDTA. Circular dichroism spectra (bottom) were recorded as described under “Experimental Procedure” at a protein con- centration of 0.82 mg per ml. Absorption spectra (top) were recorded against dialysis buffer at a protein concentration of 1.5 mg per ml.

The reactivity of modified fiZ subunit (2.9 moles of histidyl resi- dues modified per mole) was 23 to 40% of the control in all of these reactions. Although small differences in these residual activities in the various reactions were observed, these results indicate that modification affects all of the pyridoxal-P-dependent reactions of the /3~ subunit as well as the ability of the pz subunit to facilitate the indole-3-glycerol phosphate cleavage reaction of the cr subunit.

Addition of cy subunit to the PZ subunit completely inhibits its serine deaminase activity (4). The residual serine deaminase activity (23%) of the diethylpyrocarbonate-treated & subunit was also inhibited by addition of a: subunit. Four times as much (Y subunit was required to inhibit the serine deaminase activity of the control than to inhibit this activity of the modified ps sub- unit. These results show that (Y subunit does not interact with the inactive modified molecules (77% of the total) and indicate that the residual activity is due to molecules which have not been modified and which still form a normal a& complex.

York and co-workers (41, 42) have observed that the pZ sub- unit forms a fluorescent enzyme-substrate complex in the pres- ence of L-serine. The steady state concentration of this complex is reduced by the addition of NH4+ ion and even more by the addition of cy subunit. The levels of fluorescent complex formed by the control & subunit under these various conditions are shown in Table II, left column. The right column gives data for the diethylpyrocarbonate-modified pZ subunit after correction for the amount of fluorescent complex formed by the 23% of the molecules which had not been modified. These data show that modified p2 subunit does form some fluorescent comples, about

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TABLE II

Effects of NHd+ ion and cy subunit on the steady state levels oj the jluorescent L-serine complex of the fit subwit and oj the di-

ethyl pyrocarbonate-modified & subw&

I Enzyme

a Untreated 0~ subunit, diethylpyrocarbonate-treated 82 sub- unit, and fluorescence measurements are described in Fig. 6. The increase in fluorescence intensity at 500 nm in the presence of 0.1 M L-serine is expressed as a per cent of the untreated control under standard conditions.

* Since the modified enzyme contained 23% unmodified mole- cules, the observed fluorescence of the modified enzyme was cor- rected for the fluorescence of the unmodified molecules under each set of conditions by the following formula:

25y0 as much as the control, in the absence of NH4+ ion. How- ever, the steady state level of this complex was unaffected by the addition of NHd+ ion or (Y subunit.

The effects of L-serine concentration on the fluorescence in- tensity of the untreated and modified pz subunits are shown in a reciprocal plot (Fig. 6). The fiuorescence of the modified & subunit was determined after adding (Y subunit to prevent formation of the fluorescent complex by the fraction of the en- zyme which had not been modified. The apparent dissociation constant for L-serine which can be calculated from these data is 2.5 rnbf for both enzyme preparations. Thus modification seems to reduce the intensity of the fluorescence of the L-scrine complex or the amount of complex formed but does not affect it.s L-serine concentration dependence.

EJect of pN on the Maximum Velocity of the 0~ Subunit in the

Serine Deaminase Reaction-Fig. 7 shows the effect of L-serine concentration and of pH on the serine deaminase activity of the & subunit in the absence of NHh+ ion. Both the K, for L-serinc and the maximum velocity are st.rongly affected by pH. A plot of the maximum velocity versus pH is shown in the inset. The observed points fall close to a theoretical titration curve for a

Corrected fluorescence units = (observed fluorescence units group with a pK of 6.7. These values of T/Tmax are difficult to de-

- 0.23 control fluorescence units)/0.77. termine accurately since the K, for L-serine shows an approsi-

c (NH&SO4 in 0.1 M potassium phosphate, pH 7.8, was added mately loo-fold variation (from 0.05 M to 0.0005 M) over the pH

These data indicate that r,-serinc to a final concentration of 0.5 M (1 M NHd+ ion).

range tested (pH 6.1 to 8.34).

d Sufficient LY subunit was added to reduce the fluorescence of binding is dependent on at least two ionizing groups in this pH

the control to its minimal level. range.

- 400 -200 0 200 400

I/[L-SERINE]. M-’

FIG. 6. Effect of L-serine on the fluorescence intensity at 500 nm (excited at 420 nm) of the 02 subunit and of the diethylpyrocar- bonate-modified 82 subunit. The reciprocal of the increase in fluorescence intensity (l/AF) is shown as a function of the re- ciprocal of the total concentration of L-serine. The fluorescence intensity at 500 nm (excited at 420 nm) was determined before and after the addition of L-serine to solutions of 62 subunit (Prepara- tion I) in 0.1 M potassium phosphate buffer, pH 7.8, containing 1 mM dithiothreitol, 1 mM EDTA, and 0.02 mM pyridoxal-P. The untreated 82 subunit was 0.2 mg per ml. The diethylpyrocar- bonate-modified& subunit was 0.9 mg per ml. Sufficient a subunit was added to the modified @z subunit to eliminate the 23y0 residual activity due to unmodified fit subunit. The increase in fluores- cence observed on the addition of L-serine was corrected for small volume changes and normalized to that for enzyme solutions at 1 mg per ml.

I/”

0.25 025

0.20 020

0.15 0 15

0. IO 0 IO

0.05 005

I I I I I I

0 100 200 300 400 500

I I [L-SERINE] (~‘9

FIG. 7. Effect of pH and L-serine concentration on the serine deaminase activity of the p2 subunit in the absence of NHd+ ion. Reaction mixtures for the assay of the serine deaminase activity cont.ained in 1.0 ml: potassium phosphate buffer of varying pH pH (100 pmoles), pyridoxal-P (0.05 rmole), DPNH (0.1 rmole), @Z subunit (0.03 to 0.1 mg of Preparation II), lactic dehydrogenase (27 pg), and L-serine (2-200 rmoles). The decrease in absorbance at 25” was determined in a Gilford spectrophotometer for 10 to 20 min. The pH of each reaction mixture was determined at the end of the assay. The inset shows a plot of V,,, values calculated from the reciprocal plots versus pH. The solid line is a calculated theoretical titration curve for a group with a pK of 6.7.

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H+

I

‘;’ + HOCH, CCOO-

AHs+ K

+ PROTEIN-NH, H

HOCH ,+COO-

CH

H+ II

+H +

\ \

SCHEME 2

HOCH, ;COO-

1 CH

III

*OH- --

CH, * CCoo- b k!H

H+

IJL

DISCUSSIOX

Mechanism of Reaction of the pz Subunit with L-Serine-The pz subunit of tryptophan synthetase contains pyridoxal-P bound in a Schiff base linkage to an e-amino group of a lysyl residue of the protein (Intermediate I, Scheme 2) (10). The first step in the reaction with L-serine is thought to be a transaldimination step in which the amino group of L-serine displaces the amino group of the protein to form a Schiff base linkage between L-serine and pyridoxal-1 (In.termediate II, Scheme 2) (22). Recent studies (7) have assigned this structure to a fluorescent enzyme- substrate intermediate which accumulates when L-serine is added to the 0~ subunit in the absence of NI&+ ion. Abstraction of the a-proton of L-serine leads to the formation of Intermediate III. We have recently shown that the abstraction of the a-proton is the rate-limiting step in the conversion of L-serine to pyruvate in the absence of NHtf ion (7). Model (43) and enzymatic (8) studies have suggested that a histidyl or a lysyl residue may have a catalytic role in the removal of the a-proton of the substrate in pyridoxal-P-dependent transamination reactions. Trans- aminase reactions are similar to the fi elimination and 0 addition reactions of the @z subunit in that the a-C-H bond of the sub- strate is the first bond broken in all of these reactions. Mar- tinez-carrion and co-workers (44-46) have presented evidence that a histidyl residue has a catalytic role in removing the a-proton of substrates by aspartate aminotransferase. Elim- ination of the /3-hydroxyl of L-serine leads to the formation of Intermediate IV, the Schiff base of aminoacrylic acid. This intermediate can be hydrolyzed in one or more steps to form py- ruvate as shown in Scheme 2 or can undergo /3 addition reactions such asthe addition of indole to form L-tryptophan (22).

Photoinactivation of the pz Subunit-Pyridoxal-P has been previ- ously shown to be a specific photosensitizer at neutral pH for a single histidyl residue per polypeptide chain in several enzymes which bind pyridoxal-P (11-13). This is the first report to our knowledge of the use of this technique for an enzyme in which pyridoxal-P serves a catalytic role. The loss of activity which accompanies the destruction of 1 histidyl residue per monomer indicates that this histidyl residue is essential for the activity of the & subunit3 The studies of Scoffone (14, 15) suggest that a bound or covalently linked photosensitizer causes the photode- struction of adjacent amino acid residues. Thus the essential histidyl residue which is destroyed in each fi monomer is probably located adjacent to the pyridoxal-P-binding site. Although pho- toinactivation occurred when either the holoenzyme (Zntermedi- ate I, Scheme 2) or the L-serine complex (Intermediate II, Scheme 2) was illuminated, the rate of photoinactivation was four times

3 Our conclusion that a specific histidyl residue is destroyed by photo-oxidation can only be tentative in the absence of definitive data with isolated histidyl peptides. Current studies in our lab- oratory show that a single histidyl peptide containing 2 histidyl residues is largely absent after photo-oxidation. (E. W. Miles (1974) Biochem. Biophys. Res. Common., in press).

higher in the presence of L-serine. Intermediates I and II ex- hibit striking differences in their absorbance and fluorescence spectra (22, 41, 42). The IO-fold greater fluorescence of Inter- mediate II suggests that it is much less quenched due to some change in its environment. The change in the environment of pyridoxal-P might be accomplished by the rotation of the pyri- doxal-P as suggested by Ivanov and Karpeisky (47) or by some conformational change of the protein at the active site. Our finding that the rate of photoinactivation was four times faster when Intermediate II was the photosensitizer might be explained if the postulated conformational change in the formation of Intermediate II brought it closer to the essential histidyl residue. Alternatively, Intermediate II may be a better photosensitizer because its high energy state (as measured by its fluorescence) is less quenched. This high energy state is also involved in photosensitization (48).

Pyridoxal-P bound to the 0~ subunit after photoinactivation has a greater fluorescence than pyridoxal-P bound to untreated enzyme. This observation suggests that the environment of the bound pyridoxal-P is altered by the destruction of a neighboring histidyl residue. The finding that photoinactivated pz subunit retains about 70% activity in the conversion of indole-a-glycerol phosphate to indole and glyceraldehyde 3-phosphate (a reaction of the a subunit in the a& complex) indicates that photoinacti- vation does not result in a conformational change which prevents subunit interaction. However, both pyridoxal-P binding and activity in the cleavage of indole-3-glycerol phosphate by the oc&? complex are decreased 30% by photoinactivation; this sug- gests either that a certain degree of conformational change affects all molecules of /3~ subunit or that 30% of the 0% subunit mole- cules undergo some secondary modification which makes them unable to interact with coenzyme or a subunit. One such sec- ondary modification is probably oxidation of methionine to me- thionine sulfoxide since about 8 y0 of the serine deaminase activity was restored after treating the /?z subunit with P-mercaptoetha- no1 under conditions which convert methionine sulfoxide back to methionine.

Modification by Diethylpyrocarbonate-Our findings that the modification of 2 to 3 histidyl residues per /3 monomer results in inactivation and that inactivation is largely reversed by treat- ment with hydroxylamine show that 1 or more histidyl residues are essential for the activity of the pz subunit. The reduction of the rate of inactivation by pyridoxal-P suggests that one of these histidyl residues is located in the active site region or is buried in a conformation of the enzyme which is stabilized by pyridoxal- P. Modification does not greatly affect coenzyme binding or the conformation of the coenzyme binding site since the modified apo& subunit can still form a complex with pyridoxal-P which has normal absorbance, fluorescence, and circular dichroism properties. However, modification does affect interaction with the a! subunit since it results in loss of activity in the cleavage of indole-3-glycerol phosphate by the c& complex. It is clear

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that modification by diethylpyrocarbonate is less specific than modification by illumination in the presence of pyridosal-I’ and L-serine. It is probable that a mixed population of modified en- zyme molecules is obtained in which 1 to 6 histidyl residues and an unknown number of lysyl residues have been acylated. It seems likely that not all of these modified species can form the fluorescent enzyme-substrate Intermediate II (Scheme 2) which was observed in Table II and Fig. 6. Although NH*+ ion or CY subunit increase the rate of the conversion of Intermediate II to Intermediate III by untreated flz subunit and thereby reduce the steady state level of Intermediate II (42), NH4+ ion and LY subunit have no effect on the Intermediate II which is formed by the modified /LX subunit; these results suggest that modification blocks the conversion of Intermediate II to Intermediate III. This blockage could be due to some nonspecific modification of the pZ subunit or to the specific modification of a histidyl moiety which serves a specific role in the removal of the a-proton of L-serine which occurs in this step. This suggestive but incon- clusive evidence that a histidyl residue is involved in the removal of the a-proton in the rate-limiting step is supported by the find- ing reported in this paper that the maximum velocity of pyruvate formation in the absence of NH*+ ion is dependent on an ionizing group with a pK of 6.7; this pK value is typical of histidyl resi- dues but could also be due to some ionizing group of the pyri- doxal-P Schiff base intermediate.

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Edith Wilson Miles and Hidehiko Kumagail-Serine and by Diethylpyrocarbonate

Synthetase by Photo-oxidation in the Presence of Pyridoxal 5'-Phosphate and Subunit of Tryptophan2βModification of Essential Histidyl Residues of the

1974, 249:2843-2851.J. Biol. Chem. 

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