l -Phenylalanine ammonia-lyase

ARCHIVES

OF

BIOCHEMISTRY

AND

BIOPHYSICS

141,

L-Phenylalanine IV. Evidence

KENNETH Department

Group

and Mechanism

R. HANSON

of Biochemistry, New Received

(1970)

Ammonia-Lyase

that the Prosthetic Residue

1-17

AND

Contains of Action’ EVELYN

The Connecticut Agricultural Haven, Connecticut 06504 May

5, 1970;

accepted

a Dehydroalanyl

July

A. HAVIR Experiment

Station,

21, 1970

L-Phenylalanine ammonia-lyase from potato tubers upon reduction with tritiated NaBH, gave Type I tritiated enzyme. The nature of the reduction procedure, the observed correlation between loss of enzyme activity and tritium incorporation, and the magnitude of the incorporation suggested that reduction of an electrophilic center at the active site took place, and that one atom of hydrogen from borohydride is incorporated per active site reduced. Upon HCl hydrolysis, Type I tritiated enzyme gave exchangeable tritium and DLalanine-t in which most, but probably not all, of the tritium was in the p-position. It is proposed that the active site contains a dehydroalanyl residue which upon reduction yields a tritiated alanyl residue so substituted at the nitrogen atom that HCl hydrolysis leads to racemization and tritium loss. The unusual chemical environment of the alanyl residue is indicated by the mildness of the hydrolysis conditions necessary to release on-alanine-p-t and the observation that Type I tritiated enzyme upon dilution and dialysis yielded exchangeable tritium and a new form of tritiated enzyme (Type II) which gave a neutral tritiated substance upon HCI hydrolysis. Guanidine hydrochloride also brings about the Type I to Type II conversion. The enzyme functions catalytically by converting the -NHa+ of n-phenylalanine into a better leaving group. If the nitrogen of the dehydroalanyl residue is present as a Schiff’s base masked against attack by such reagents as borohydride, then it is probable that the amino group of phenylalanine adds to the P-position of the dehydroalanyl double bond. If this intermediate undergoes a prototropic shift the conjugated system O=C-C*=Crr-N may be formed (the three carbon atoms are those of the original dehydroalanyl residue and the nitrogen belongs to phenylalanine). With such a leaving group the free energy of the transition state in the elimination process should be lowered by resonance.

n-Phenylalanine ammonia-lyase (EC 4.1. 3.5) catalyzes the elimination of ammonia from n-phenylalanine to give trans-cinnamate and ammonium ions (1). In studies of the enzyme purified from light-exposed slices of potato tubers (2, 3) kinetic evidence was obtained for the formation during the catalytic sequence of an enzyme-ammonia 1 This investigation Grant GB-12428 from dation.

intermediate. This suggested that, once phenylalanine is bound to the enzyme, an electrophilic center at the active site is able to combine with the nitrogen of the substrate to form a group with better leaving properThis intermediate should ties than -NH,+. undergo an elimination reaction to yield enzyme-bound cinnamate. The cycle is completed by release of cinnamate and hydrolysis of the remaining amino- (or imino-) enzyme intermediate.

was supported in part by the National Science Foun1

Copyright

@ 1970 by Academic

Press,

Inc.

2

HANSON

In experiments directed toward the identification of the electrophilic center, it was found that the enzyme could be reversibly inhibited by such carbonyl reagents as phenylhydrazine, semicarbazide, and cyanide, and irreversibly inactivated by NaBH4. L-Phenylalanine, cinnamate, phenylhydrazine, and semicarbazide all protected the enzyme against inactivat’ion by NaBH4 . As cinnamate gives t’otal protection of the electrophilic center against NaBH4 attack, it is unlikely that any random release of products takes place in the catalytic sequence. Both L-phenylalanine ammonialyase from Rhodotorula qlutinis (4) and Lhistidine ammonia-lyase from a Pseudomonas species (5, 6) show similar carbonyllike properties. Our further investigations employing tritiated NaBH4 (hereafter written NaBH4t4) led us to suggest that a dehydroalanyl residue is present at, the active site (7,8). We here report in detail the evidence for this assertion: namely that tritiation of the enzyme with NaBH& followed by dialysis as a concentrated solution and HCl hydrolysis led to the release of m-alanine labeled primarily in the methyl group and of exchangeable tritium. Such preparations we now called Type I tritiated enzyme. We further report that Type I tritiated enzyme after dilution and dialysis, or after denaturation with guanidine hydrochloride, no longer gives tritiated alanine but an unidentified neutral substance, Compound B. In discussing the way in which a dehydroalanyl residue might function as part of a prosthetic group we shall take into account recent studies on hi&dine ammonia-lyase (9, 10). Most of our experiments have been carried out with purified potato enzyme (2, 3, ll), but reference will be made to parallel studies of the enzyme from maize (12) to be reported more fully elsewhere (13). METHODS Protein hydrolysis. All protein hydrolyses were performed with degassed solutions in tubes evacuated to 20 mm Hg and sealed (14). Unless otherwise indicated, acid hydrolyses were carried out in 6 N HCl at 110” for 18 hr. The product was separated into residue and sublimate with the aid of

AND

HAVIR

n

FIG. 1. Apparatus for vacuum sublimation. The apparatus was blown and assembled from parts obtainable from the Kontes Glass Co., Vineland, N.J., namely, a solid high vacuum stopcock with stopcock retainer, pinch clamps, connector tubes 15-mm i.d. taking size 116 Type M O-rings, and test tubes taking similar rings and sold for use with a vacuum hydrolysis assembly (10, 25, or 46 ml). Tapered flasks were blown from such tubes. Provided that the O-rings were wiped with a trace of silicone grease and the stopcock retainer tightened, a high vacuum in the U-assembly held for hours or days. The apparatus, which has been used for the lyophylization of enzymes, the removal of aqueous HCI, and the removal of volatile organic reagents, has proved to have many advantages over earlier devices (15).

the vacuum sublimation apparatus shown in Fig. 1, and the distribution and recovery of radioactivity were determined. For convenience the term “sublimate” is used in this case and in others where both sublimation and distillation took place. Zone electrophoresis on paper. Separations were performed on Whatmann 3MM paper with an EC electrophoresis apparatus (EC Corp., Philadelphia, Pa.) at 15” and a potential gradient of 15 to 20 V/cm, or with a Savant (Hicksville, N.Y.) 45-cm flat-plate system at 10” and 54 to 62 V/cm. The buffers employed were: pH 1.9, formic acidacetic acid (60 ml + 240 ml to 2 liters); pH 3.5, pyridine-acetic acid (6.6 ml + 66 ml to 2 liters); pH 5.7, pyridine-acetic acid (20 ml + 8 ml to 2 liters); pH 5.9, KH phthalate-NaOH (0.025 M); pH 7.4, NaHzPOa-Na,HPO1 (0.02 M); pH 9.1,

DEHYDROALANINE

IN

PHENYLALANINE

Na2BnO?-H,B03 (0.02 M calcd as borate); pH 10.3 and 10.7, Na2C03-NaHC03 (0.02 M). Gas-liquid partition chromatography. Separations were performed on a Perkin-Elmer 810 gas chromatograph attached to a Barber Colman combustion furnace and radioactivity monitor. The SE-30 column for separating trimethylsilyl amino acids consisted of 5% silicone gum rubber on Anakrom SD 9&100 mesh (both from Analabs, North Haven, Conn.) in a 120.cm coiled copper tube x in. od. Conditions: column, 125”, injector, 225”; transfer heater for combustion furnace, 170”; argon carrier gas, 75 ml/min; conditioning, 240” for 20 hr. The EGA column for separating 0-butyl N-trifluoroacetyl amino acids consisted of 2% ethylene glycol adipate on silicone-treated Chromosorb W 80-100 mesh (both from Analabs) in a lOO-cm stainless-steel tube K in. o.d. Conditions: column, 107”, injector, 185”; transfer heater, 170”; argon, 100 ml/mm; conditioning, 235” for 20 hr. Exchangeable tritium by (NH&SOa transfer. Samples of water were examined for the presence of avolatile tritiated contaminant as follows. Each sample was diluted to about 2.5 ml and portions (3 X 100 ~1) were withdrawn for scintillation counting. Crystalline (NHa)&Od (1.7 g) was dissolved in 2 ml of the diluted sample at 100”. The solution was cooled in ice and stirred until small crystals were deposited. These were collected by filtration, washed with acetone and ether, then weighed (ca. 0.3 g), dissolved in water (200 J/ 100 mg), and portions (3 X 100 ~1) were withdrawn for scintillation counting. In all assays triplicate determinations with a stock solution of tritiated water (100 gl = 1360 cpm) were carried out in parallel with the sample determinations. The percentage of the tritium in the original sample that was exchangeable = 100 (70transfer for sample)/ (% transfer for tritiated water). An analysis of variance for the transfer of tritium from tritiated water in six experiments of three replicates each (mean transfer 14.7%) showed that in a given experiment the standard error (estimate of standard deviation) of individual observations was 0.4y0 (coefficient of variation 2.87,). The greater part of the overall scatter of observations arose from day-to-day variations in procedure. The products from the HCl hydrolysis of tritiated protein were examined by first making the acid sublimate alkaline with solid NaOH and then performing a further sublimation. The exchange assay was carried out on this neutral sublimate. Water was added to the alkaline residue and then removed by sublimation. After this step the residue contained less than 570 (usually about 1%) of the tritium of the acid sublimate. Thus, the acid sublimates did not contain significant amounts of

AMMONIA-LYASE

3

volatile trit.iated acids (e.g., acetic-t acid). Accurate assays of the neutral sublimate could be obtained with as little as 200 cpm/lOO ~1 of diluted sample, i.e., 5090 cpm total. As the percentage of tritium of each neutral sublimate examined was calculated to be in the range 94-108% exchangeable, specific values have been omitted from the Results section. Radioactivity in alanine by isotopic dilution. A large excess of unlabeled on-alanine (5pmoles) was added to the hydrolysis residue or other sample to be examined, and a portion of the alanine separated by descending chromatography on Whatmann 3MM paper with 1-butanol-acetic acidwater (4:1:5 by vol, upper phase). The radioactivity in the alanine of the original sample was calculated from the specific activity (e.g., 300 cpm/Fmole) of the eluted alanine which was assayed with ninhydrin-hydrindantin (16). Corrections were made for ninhydrin-positive material in the paper, and the procedure was checked by chromatographing alanine-t of known specific activity on the same sheet of paper. Specijk activity of glycolate-2-t. In order to estimate the extent of hydrogen incorporation into tritiated enzyme, a sample (about 1 rmole) of the alkaline solution of NaBHa-tr (New England Nuclear) employed to reduce the enzyme was added at 0” to a solution of Na glyoxylate (4 pmole). Exchangeable tritium, possibly derived from contaminants in the NaBH& , was removed by sublimation (about 50% of the tritium in the solution). Two columns (2 X 0.5 cm) of Dowex l-X8 acetate in Pasteur pipets were washed with 10 N acetic acid (8 ml) and water (8 ml). A portion of the residue (about 0.2 pmoles glycolate-2-t) was applied to the sample column, and the sample and blank columns were developed with water (4 X 1 ml) and 4 N acetic acid (10 X 0.5 ml). Glycolate was determined (17, 18) with 2,7-dihydroxynaphthalene in coned H,SOd , and a correction was made for the blank readings. About 90% of the applied radioactivity was associated with the glycolate peak which showed a drop in specific activity from front to back. Duplicate determinations upon the same sample gave satisfactory agreemeut between the mean specific activities of the glycolate-Z-t effluent peaks. The specific activity of samples of glycolate-2-t determined after descending paper chromatography in l-butanol-waterethanol-diethylamine (80: 20: 10: 1 by vol) (19)) and ether-acetic acid-water (13:3:1 by ~01) (20) were similar to those assayed as above, but as the paper yielded compounds that gave appreciable color in the glycolate assay this method was abandoned. Tritium distribution in alanine-t. The method of Gilvarg and Bloch (21) for determining the dis-

4

HANSON

tribution of 1% in alanine was adapted. Alanine (20 pmole), KMn04 (6 mg), H,POa (60 pl), and water were added to a sample of alanine-t (800 to 56@0 cpm); final vol 800 ~1. The solution, in a 25ml sublimation tube (see Fig. 1) covered with a glass bulb having a short stem that sat in the mouth of the tube, was heated at 95” for 40 min, cooled, and the remaining KMnOa reduced with 37% formaldehyde (10 ~1). The exchangeable tritium and acetic-t acid formed were removed by sublimation. Samples of the sublimate were taken for scintillation counting, a further sample (400 ~1) was made alkaline with 4 N NaOH (25 J), and the exchangeable tritium was separated from sodium acetate by sublimation. Water (425 ~1) was added to the residue, and the radioactivity in lOO+l aliquots of the residue (T) and the neutral sublimate (s) was determined. The recovery of activity calculated for this step was essentially quantitative. The minimum percentage of tritium in the @-position of alanine-t = 100 T/(T + s). Between 2 and 10% of the tritium of the alanine-t sample remained in the syrupy phosphoric acid residue. Only a portion of this radioactivity could be removed by adding water and repeating the sublimation. As repeated determinations showed no correlation between the estimated amount of p-tritium and the amount of residual tritium, the residue was ignored in calculations of @-tritium. Samples of nL-alanine-8-t were prepared from L-alanine-U-t (Amersham-Searle) by heating degassed solutions of the amino acid (20 mg) with CuSOa (4 mg) and salicylaldehyde in water (300 pl) for 76 or 124 hr at 110” in evacuated sealed tubes (22). Roughly one-third of the recovered tritium was exchangeable in both cases. Portions of the product were purified by descending paper chromatography in 1-butanol-acetic acid-water (4:1:5 by vol, upper phase). Material from the front and back of the alanine region of the chromatogram in each case was assayed for @-tritium in a single experiment: 76 hr, 94 and 93%; 124 hr, 93 and 95.6%. We conclude that an assay value of about 94% fl-tritium corresponds to 100% /3-tritium in the sample. The assay value for fl-tritium in alanine-t purified from a single hydrolyzate of Type I tritiated enzyme (see Results) varied from 80-92$& (seven separate experiments, mean and standard error of mean, 86 f 1.7%; sample SE 4.4%) thus scatter from experiment to experiment was appreciably greater than that of replicate values in a single experiment. As no outlying values have been observed, and as a determination upon a standard sample of alanine-t was always run in parallel with other determinations, the low values for @-tritium obtained with certain samples were not accidental. Preparation of Type I tritiated enzyme. Enzyme

AND

HAVIR

(0.8 ml, 20 mg protein/ml) from potatoes, purified in the final stage by chromatography on an Agarose column (Bio-Gel A-l.5 M, 200-400 mesh; from Bio-Rad) to a specific activity of 585 mU/mg protein (II), was freed from (NHJzSOa and transferred to 0.05 M phosphate (K+) buffer, pH 6.8, by passage t,hrough a column (7 X 0.5 cm) of Sephadex G-25 (Pharmacia) in a disposable Pasteur pipet. The fractions containing most of the enzyme were combined (6.73 mg protein/ml, 1.5 ml) and 2 pmoles of cinnamate added (final concentration 1.2 mM). Two portions of NaBH4 solution were added at 0” to a final concentration of 0.1 mM. The reaction was complete in less than 5 min (3). The enzyme showed no loss of activity on assay whereas in a control reaction, which contained borohydride but no cinnamate, the enzyme was completely inactivated. The remainder of the reaction mixture was dialyzed for 2 hr against a liter of 0.05 M phosphate buffer, pH 6.8, to remove the cinnamate. The specific activity of the enzyme was unchanged after dialysis. A solution of NaBHa-ta (New England Nuclear, e.g., 30 mCi/ 0.15 mg) was prepared at 0” in 0.1 N NaOH (200 ~1). After each lo-p1 addition of this solution to the reaction mixture at O”, a sample (50 ~1) was removed for assay. After the fourth addition, less than 10% of the original enzyme activity remained. The reaction mixture was dialyzed (18 hr, 4 X 2 liters) against 0.05 M phosphate buffer, pH 6.8. The dialyzate did not contain appreciable radioactivity after the third change of buffer. The reduced enzyme w&s assayed for protein (23) and radioactivity. Samples of this Type I tritiated enzyme which had been stored at - 20” for some time did not completely dissolve upon thawing, but upon HCl hydrolysis they gave the same tritiated product (Compound A, see Results) as freshly reduced enzyme. Proteolysis of Type I and Type ZZ tritiated enzyme. (a). To Type I tritiated enzyme (0.18 mg protein; 849,000 cpm) in 0.05 M (NH4)&03 was added trypsin free of chymotryptic activity (0.1 mg, 265 (TAME) U/mg; enzyme treated with L-(tosylamido-2-phenyl)-ethyl chloromethyl ketone from Worthington), final vol 0.2 ml, and the solution was maintained at 23” for 16 hr. Pronase (0.2 mg; 45,000 (PUK) U/mg; B grade from Calbiochem) was added, and after 24 hr the solution was heated for 3 min at 100”. The slight precipitate was removed by centrifugation and the entire supernatant fraction subjected to paper electrophoresis at pH 1.9 and 3000 V for 1 hr. The major portion of the radioactivity elutable with water was located at the origin, but a peak of radioactivity (5000 cpm) W&S associated with the alanine region. (b). Type I tritiated enzyme (1.36 mg protein; 488,000 cpm) was converted to denatured Type II

DEHYDROALANINE

IN

PHENYLALANINE

tritiated enzyme by treatment for 30 min at 23” with guanidine hydrochloride (2.3 g), Tris (24 mg), and EDTA (5 mg) ; final vol, 4 ml. During this time the pH was adjusted to 8.5 with 1 N NaOH by means of a pH Stat (Radiometer). Mercaptoethanol (5 ~1) was then added. After 1.5 hr at pH 8.5, iodoacetic acid (15 mg) was added, and finally, after 1 hr at pH 8.5, the reaction mixture was dialyzed overnight against 1 liter of water. A sample was removed for HCl hydrolysis, and the protein (1.32 mg, 278,000 cpm) was maintained at pH 9.0 with 0.1 N NaOH during the addition over a 3-hr period of trypsin free of chymotryptic activity (3 X 0.1 mg) and then overnight. A sample was removed for HCI hydrolysis and the pH maintained at 8.4 during the addition of pronase (2 X 0.1 mg) and then overnight. Finally, the reaction mixture was chromatographed on a Sephadex G-50 column (42 X 2.5 cm). About 90% of the radioactivity was recovered in the low molecular weight “amino acid” fraction and found to be associated with Compound B (see Results) upon examination by electrophoresis and thin-layer chromatography. The samples subjected to HCl hydrolysis gave exchangeable tritium and Compound B. RESULTS

Preparation of Enzyme SpeciJically at the Active Site

Tritiated

Type I tritiated enzyme. When samples of potato phenylalanine ammonia-lyase, with specific activities greater than two-thirds the highest observed, were reduced and inactivated with NaBH& , tritium was incorporated into the enzyme. It seemed probable that some of the tritiated products obtained from this material by acid hydrolysis arose through the action of NaBH& upon portions of the enzyme remote from the active site and upon contaminating proteins. A standard procedure was, therefore, developed (see Methods) in which the enzyme was first reduced at 0” and pH 6.8 with approximately four times the minimal amount of unlabeled NaBH4 necessary to inactivate the enzyme and in the presence of sufficient Na cinnamate to protect it completely against inactivation (3). The enzyme was then separated from cinnamate, etc. by dialysis and reduced with the minimal amount of NaBH4-t4 required for inactivation. As it is not possible to prepare accurately a stock solution of NaBH4-t4 of

AMMONIA-LYASE

5

known concentration, the amount necessary for this step had to be determined either by adding successive portions of NaBH,-t, solution and following the loss of activity, or by carrying out several small-scale preliminary reductions. Exchangeable tritium was removed by exhaustive dialysis against pH 6.8 phosphate buffer. As will be explained below, it proved to be important to maintain a relatively high protein concentration in the dialysis tubing (2-3 mg/ml). Samples of tritiated enzyme obtained in this way were hydrolyzed with 6 N HCl under standard conditions (llO”, 18 hr). Close to 50% of the radioactivity was present in Ohe sublimate. The same result was obtained with tritiated enzyme from seven different reduction experiments performed upon three different batches of purified enzyme. All of the tritium in the sublimate was freely exchangeable (four samples), and upon examination of the residue by electrophoresis (Fig. 2) only one significant peak of radioactivity (Compound A) could be detected either directly or after elution. Once Compound A had been identified as ala&e-t (see below), it was shown by isotopic dilution that at least 90 % of the radioactivity in the residue was associated with alanine. All such batches of tritiated enzyme that yielded alanine-t upon acid hydrolysis we shall call Type I tritiated enzyme.

If the NaBH4-cinnamate step was omitted from the reduction procedure and the tritiated enzyme obtained was hydrolyzed, the amount of alanine-t released varied. In one experiment more than 80% of the tritiated material in the residue from hydrolysis was alanine-t whereas in another it was about 66 %. All batches of enzyme that gave alanine-t by the standard reduction-hydrolysis procedure gave alanine-t as the major product when the protection step was omitted. As the standard reduction procedure is relatively wasteful of enzyme, the maize enzyme (sp act 97 mU/mg> was reduced with the minimal amount of NaBH4t4 necessary to cause inactivation. Approximately 60% of the tritium in the residue after acid hydrolysis was in alanine-t. In a control experiment, performed at t’he

HANSON

AND

HAVIR

600

pH 1.9

Ah

Gly

Aq

-t

pH 10.7

n

600400zooo-

+$o

0

-

-

-L-z-

+io MIGRATION

’

,4

0

(cm)

FIG. 2. Electrophoretic examination of products from the HCl hydrolysis of the three types of tritiated enzyme. Narrow strips cut from the paper after electrophoresis were added to vials of scintillator solution for the determination of radioactivity. Upper panel: Sample of the residue from the HCl hydrolysis of Type I tritiated enzyme prepared by the standard procedure (see Methods). Compound A is identical with alanine-t (see Results). In the control experiment cinnamate was omitted from the NaBH4 reduction step so that the enzyme was completely inactivated before being treated with NaBH,-t, . Middle panel: Sample of Compound B from the hydrolysis of Type II tritiated enzyme and a sample of the mixture of compounds (B’) accompanying Compound B when certain samples of Type I tritiated enzyme that had been denatured with guanidine hydrochloride were hydrolyzed. Lower panel: basic tritiated L-amino acid obtained upon the HCl hydrolysis of certain batches of tritiated enzyme.

same time as a standard reduction and with the same reagents, enzyme was treated with NaBH4 in the absence of cinnamate and then with the amount of NaBH& required to inactivate the enzyme. About 10% as much tritium was incorporated and a negligible amount of this t’ritium remained in the residue after HCI hydrolysis (Fig. 2, control electrophoresis). It follows that the tritium in the Type I tritiated enzyme must have

been incorporated into a region of the protein which is not accessible to the reagent in the presence of cinnamate. In the standard reduction procedure, the enzyme is separated from any (NH&S04 present in the stock solution by Sephadex G-25 chromatography prior to the NaBH4cinnamate step and dialyzed or, in some experiments, passed through a second Sephadex G-25 column before reduction

DEHYDROALANINE

IN

PHENYLALANINE

with NaBHp-t4. It is very unlikely, therefore, that an amino or imino enzyme intermediate in the reaction sequence and not the free enzyme was being reduced. The enzyme was not protected against NaBH4 inactlvation by 0.8 mM (NH&Sob, and this concentration of (NH&SO4 did not alter the protective action of cinnamate or L-phenylalanine. When a large excess of unlabeled NaBH4 was added to Type I tritiated enzyme, the product upon HCI hydrolysis gave exchangeable tritium and alanine-t in the rat’io previously observed. Table I lists estimates of the number of atoms of borohydride-derived hydrogen incorporated per molecule of Type I tritiated enzyme. Glyoxylate reduction to glycolate2-t was employed as a reference. The variability of the results indicates that isotope effects occur. As all four hydrogens of NaBH4 are available for reduction, four reagents rather than one were being employed and any factor changing the detailed kinetics of reduction and hydrolysis may have influenced the relative amounts of tritium incorporated into glycolate and enzyme. Tritiation does not permit a meaningful calculation of t.he number of active sites per molecule, but it is reasonable to postulate that at least one atom of borohydride-derived hydrogen is incorporated per active site reduced. One major batch and two small samples of purified enzyme reduced w-ithout any prior cinnamate-NaBH, step gave on HCl hydrolysis about 20 % exchangeable tritium and a residue containing as t’he major product a strongly basic L-amino acid (Fig. 2, Compound C). This compound resembled but was not ident,ical with arginine.2 As 2 Compound C and six compounds obtained by its chemical modification have been characterized by electrophoresis in various buffers and by t,hinlayer chromatography in three solvent systems. We have been unable to discover whether the strongly basic group, which does not react with HNO:! , is a substituted guanidinium group or a quaternary ammonium (as in c-N-trimethyl-Llysine (24)). When the a-carbon of Compound C was converted to COOH with the aid of L-amino acid oxidase and Hz02 and the butyl ester of this treated with acetic anhydride for 3 min at 130”, a neutral ester was obtained. Although this reaction

7

AMMONIA-LYASE TABLE

I

EXTENT OF INCORPORATION OF BOROHYDRIDEDERIVED HYDROGEN INTO TYPE I AND TYPE II TRITIATED ENZYME

Expt.’

sp+ic “;;;$y reduction (mU/mg protein)

Type of tntmted enzyme prod”ced

Calculated incorporation bprohydridedey;;tzF

Observed tritium incorporation Into glycolate-Z-t (103 cpm/ nmole)

Into enzyme

of

“‘CO rected

CP’;twt;d a

q2glg protein)

k$zlTe enzyme)

e;;Fz$

225 149 500 223 206

0.75 0.20 2.1 0.9 0.46

-

1 2 3 4

648 585 585 585 450

I I I II I

100 250 80 80 150

0.97 0.29 3.00 1.28 0.85

n Three different batches of enzyme were reduced and inactivated with NaBH& to yield Type I tritiated enzyme according to the standard procedure (see Methods). Experiments 2 and 3 were performed on the same batch of purified enzyme. The sample of Type II tritiated enzyme (Expt. 3) was obtained by dialyzing the reaction mixture under the conditions employed to prepare Type I tritiated enzyme except that the protein concentration was 0.09 mg/ml instead of 3 mg/ml (see Fig. 3). In each experiment a portion of the NaBH1-tr used to reduce the enzyme was also used to reduce a sample of glyoxylate to glycolate2-t and the specific activity of this product was determined (see Methods). The amount of borohydride-derived hydrogen incorporated into the enzyme may then be calculated if it is assumed that the kinetic isotope effects for the reduction of the enzyme are the same as for the reduction of glyoxylate. The “uncorrected” incorporation is calculated on the further assumptions that the molecular weight (2) of the enzyme is 330,000, that the weight of protein is correctly estimated by the method of Lowry (23), and that all of the protein is enzyme. These figures are corrected (the last column) on the assumption that the highest specific acbivity observed during purification experiments, namely 835 mU/mg protein, is that of the pure enzyme.

only a small amount of alanine-t was present, and as the amount of tritium incorporated is reminiscent of the first step in the BergmannKoster degradation of arginine to ornithine (25, 26), the neutral ester on HCl-hydrolysis gave an acid lacking any basic function. The results of hydrolysis experiments with Ba(OH)2 were also inconclusive (27).

8

HANSON

AND

into the protein was comparable to the amounts observed with Type I tritiated enzyme, it seems probable t.hat subtle changes in the conformation of the protein arising on storage allow t,he active site to undergo an alternative type of reduction. This result has not been repeated with recent batches of purified enzyme, and Compound C has not been observed as a trace product accompanying alanine-t. Type II tritiated enzyme. When samplesof Type I t,ritiated enzyme were diluted immediately after reduction to ca. 0.09 mg protein/ml and dialyzed against buffer as before, the product on HCl hydrolysis yielded about 50 % of its radioactivity as exchangeable tritium (four samples) and t’he residue upon electrophoresis appeared to contain a single neutral tritiated product: Compound B. The properties of t’his compound are discussed further below. All samples of tritiated enzyme giving Compound B we shall call Type II tritiated enzyme. No transformation upon dialysis took place wit,h stored Type I tritiated enzyme. Close to 50 % of the tritium is lost from the protein during the Type I to Type II transformation as determined both by comparing the total radioactivity in the tubing before and after dialysis and by comparing specific activity values (Table I, Expt. 3). When samples of enzyme partially reduced with NaBH& were dialyzed as dilute solutions, the loss of enzyme activity upon reduction was proportional to the amount of tritium

incorporated

into Type II tritiated

enzyme (Fig. 3). Presumably, had it been reasonable to employ larger amounts of enzvme for the experiment, the same proportionality would have been observed for incorporation into Type I tritiated enzyme. If two reductions had taken place in the

tritiation process, the one reduction inactivating the enzyme and the second reduction attacking another part of the active site at a significantly different rate, then proportion-

ality need not have been observed and would not have been observed if the two reductions were independent.

The results

are consistent

with the view that inactivation and tritiation are one and the same process.

HAVIR

, 100.000

0 INCORPORATED

RADIOACTIVITY

I\ 200.000 (cpm/mg

I protem)

FIG. 3. Proportionality between the loss of enzyme activity upon reduction and the incorporation of tritium into Type II tritiated enzyme. The enzyme was reduced with NaBH& according to the standard procedure (see Methods) and the dilute mixtures (3 ml, 0.09 mg protein/ml) employed in assaying the enzyme for activity after each successive addition of NaBH& were dialyzed against phosphate buffer and then assayed for protein (23) and radioactivity. The highest incorporation value shown is also recorded in Table I (Expt. 3).

Investigations of Compound A and Type I Tritiated Enzyme IdentiJication of Compound A as DL-Alanine-t. Compound A, derived from Type I tritiated enzyme by HCl hydrolysis, and several of its derivatives were examined by electrophoretic and partition methods. Where appropriate, sufficient alanine or its derivative was added to permit the location of the carrier (detected by ninhydrin color, fluorescence, etc.) to be correlated with the distribution of radioactivity. (a). Elect’rophoresis established that Compound A was uncharged at pH 5.7, had a charge of - 1 at pH 10.7, and at pH 1.9 had a fractional positive charge such that it migrated with alanine (Fig. 2). (b). On thin-layer chromatography in acidic and basic solvent systems the single peak of radioactivity was associated with the alanine region, e.g., 1-butanol-acetic acid-water (4: 1: 1 by vol) and l-propanol34 % NH,OH (7: 3 by vol) : separations on ChromAR Sheet-500 (Mallinkrodt). In both

DEHYDROALANINE

IN

PHENYLALANINE

TABLE

TAB-DERIVATIVES

n

ACTION

wb L-C L- then

1L

0

5

IO TIME

15

(MINI

FIG. 4. Identity in behavior upon gas-liquid partition chromatography of the 0-butyl N-trifluoroacetyl (TAB) derivatives of Compound A and alanine. The derivative of Compound A was cochromatographed at 107” on a 2% ethylene glyco1 adipate (EGA) column (see Methods) with the mixture of TAB amino acids indicated: smooth line, flame ionization detector; irregular line, radioactivity monitor. The mass of the TAB-Compound-A sample alone was too small to produce a response by the flame ionization detector. The control run at 106” shows the offset in t.ime between the mass and radioactivity records. In this experiment TAB-Ala-r4C and TAB-Gly-r4C were cochromatographed with the same mixture of TAB amino acids. The difference in temperature between the two runs arose because of difficulty in maintaining low constant temperatures when the combustion furnace was operating. Reference TAB-amino acids were prepared as described by Lamkin and Gehrke (31).

systems alanine was resolved from serine and glycine; Rp values were about 0.2 greater than on Silica Gel G (28). The radioactivity was also associated with the alanine region after descending chromatography on Whatmann 3MM paper in lbutanol-acetic acid-water (4 : 1: 5 by vol, upper phase) and 2-butanol-3 % NH,OH (3:l by vol). (c). The trimethylsilyl (TMS) derivative of Compound A was prepared with bis(trimethylsilyl)acetamide (29) and examined by gas-liquid chromatography on a non-

II

OF D- AND L-AMINO ACID OXIDASES COMPOUND A (ALANINE-t)a

Amino acid oxidase

CONTROL

9

AMMONIA-LYASE

D-~

ON

Remainingalanine-t after treatment of samplesfrom tritiated enzyme Potato Type I Maize tritfated tritiated enzyme enzyme (%) (%) 48 51 1

61 51 12

a The samples of alanine-t from the HCl hydrolysis of tritiated enzyme were purified by paper chromatography. The sample from Type I tritiated potato enzyme corresponds to the sample of line 2, Table III. After treating the samples with amino acid oxidase as indicated below the reaction mixture was transferred to a Dowex 50- X8 H+ column (2 X 0.5 cm) in a Pasteur pipet, eluted with water (4 X 0.4 ml) to remove exchangeable tritium and pyruvate, and then with 2 N NH,OH (4 X 0.4 ml) to remove residual alanine-t. Applied and recovered radioactivities were in satisfactory agreement, but the fraction of alanine-t remaining was calculated as a percentage of the total recovered radioactivity. 6 Reaction mixture: sample, ca. 2000 cpm; pyrophosphate (Na+) buffer, pH 8.2, 1 pmole; n-amino acid oxidase from hog kidney (purified electrophoretically, 2.9 U/mg; from Sigma) 50 rg; final vol 50 ~1. The solution was left for 3 hr at 23”. c Reaction mixture: sample as above; Tris (Cl-) buffer, pH 7.2,2 pmole; L-amino acid oxidase from Crotalus adamanteus (5.4 U/mg; Type IV from Sigma), 25 pg; final vol 50 ~1. The solution was left for 3 hr at 23”. d The reaction mixture as (c) was left for 3 hr at 23”, then 50 pg of n-amino acid oxidase were added and the solution left for a further 3 hr before fractionating. A similar experiment was performed on samples of Compound A (3000 cpm) in which the amount of residual alanine-t was determined by isotopic dilut,ion. After the reaction mixtures had been left for the times indicated, 5 pmoles of alanine were added to each solution to give final volumes of 0.3 ml. The reaction mixtures were heated to coagulate the enzyme and in each case alanine-t was isolated and its specific activity determined (see Methods). The residual alanine-t was for the potato enzyme sample: D, 50; L, 45; D then L 1.5%, and for the tritiated maize enzyme sample: D, 55; L, 48%.

polar column (5% SE-30) along with TMS derivatives of other amino acids. Working at the lowest column temperature practicable

10

HANSON

(125”), the retention time for the radioactive compound was about 1.7 min; similar to the times for TMS-alanine and TMS-glycine and significantly less than those for other amino acids (e.g., TVS-valine, 3.25 mm). (d). The 0-butyl N-trifluoroacetyl (TAB) derivative (30) of Compound A was examined by gas-liquid chromatography on a polar column (2% EGA) along with other TABamino acids. The peak for the radioactive derivative (5.3 min) exactly coincided with the mass peak for TAB-alanine (Fig. 4) and differed in retention time from the peaks for such related compounds as TAB-a-aminobutyrate (6.2 min) and TAB-p-alanine (13 min). In general, gas-liquid partition coefficients with nonpolar columns are less sensitive to structural differences than with polar columns, thus the two gas-liquid chromatography procedures are complementary and not measurements of the same effect’s upon partition. (e). The 5-dimethylamino-l-napthalenesulfonyl (“dansyl”) derivative (32, 33) of Compound A was prepared by treating the residue from the HCI hydrolysis of Type I tritiated enzyme (10,000 cpm; less than 1 pg-atom amino N) with dansyl chloride (5 mM) in XaHC03 (0.1 M) at 25”; final vol 0.2 ml. The single radioactive product migrated at the same rate as carrier dansylalanine when examined by electrophoresis at pH 1.9 and thin-layer chromat)ography on silica gel G in 1-butanol-acetic acidwater (4: 1: 1 by vol). (f). Upon treatment of Compound A with nitrous acid (NaN02, 2 M; H,SO, , 1 N; final ~010.1 ml) and chromatography of the product on Whatman 3R!IM paper in ether-acetic acid-water (13 : 3 : 1 by vol; ascending) (20), 85 % of the detectable radioactivity was present in the lactic acid region (Rp 0.7). D:L Ratio and position of labeling. The D:L ratio for the alanine-t isolated from samples of tritiated potato and maize enzymes was determined with the aid of nand n-amino acid oxidases (Table II). As the release of trit’ium from the products oi amino acid oxidase action depends upon the experimental conditions as well as the position of labeling (34), t’he only satisfactory

AND

HAVIR

method was to assay the amounts of alanineremaining after prolonged treatment with the enzymes. In the experiments recorded, and in various preliminary experiments which examined alanine-t from different batches of tritiated enzyme, the amino acid appeared to be racemic. The position of tritium labeling was investigated by oxidizing samples of isolated alanine-t to acetic-t acid and exchangeable tritium with acidic permanganate (Table III). All tritium in the a-position becomes exchangeable, and when authentic alanine-p-t was oxidized, 94% of the tritium remained in t’he methyl group of acetic acid. If bhis value is taken as a standard, then about 90 % of the tritium in the major batches of alanine-t prepared from Type I tritiated enzyme was present as alanine-p-t (lines 2 and 3). Although there is sufficient statistical uncertainty in t’he oxidation procedure for the true values for these samples to be 100% p-tritium, the results of lines 4 and 6 cannot be attributed to statistical error. Either these samples were grossly contaminated with an unknown compound giving tritiated water upon oxidation, or tritium was present in the a-position. In the case of the sample from t’he tritiated maize enzyme (the only sample from maize large enough to allow an oxidation experiment), electrophoresis at pH 1.9 showed only one peak and that corresponded to the alanine region. No radioactivity was associated with this region when the sample was treated with nitrous acid before electrophoresis. It is probable, therefore, that the original tritiated protein contains some alanyl residues with cY-tritium and some with p-tritium and that t’ritium is normally lost from the (Yposition upon hydrolysis. Such losses would be accompanied by racemization. Unfortunately, we do not know how to reproducibly manipulate the enzyme in such a way that ac-tritium is retained. HCl hydrolysis. In addition to the hydrolysis experiments employing 6 N HCl for 18 hr at’ 110”, milder conditions for the hydrolysis of Type I tritiated enzyme were investigated, e.g., treatment at 110” with 6 N HCl for 1 hr (see also Table III) and 0.03 N HCl for 16 hr yielded respectively 75 and 50 % of t

DEHYDROALANINE

IN

PHENYLALANINE TABLE

DISTRIBUTION

Limb

5 6 7 8

OF TRITIUM Source

IN ALANINE-t

III

DETERMINED

of alanine-lc

oL-Alanine-p-t (chemically prepared) Type I tritiated potato enzyme As 1, but different reduction Same reduction as 1, but stored tritiated enzyme Same reduction as 1, but trypsin and pronase digestion Tritiated maize enzyme Tritiated nisind As 7, but two other reduction+

11

AMMONIA-LYASE

BY ACID

Time of HCI hydrolysis

18 1, 4, 1 1, 4,

18 18 18

(hr)

18 18

PERMANGANATE No. of observations

4 7 1 each 1 1 each 1

OXID.~TION~ Minimum

% of tritium

in @-position

87, 50,

2 2 4

94 86 89, 34 61, 81

86 52

60 40 77

a Principle of assay. Upon oxidizing alanine to acetic acid with acid permanganate (see Methods) TCHICHNH&OOH gives TCHZCOOH and CH&TNH#OOH gives exchangeable tritium. As some acetic acid may be further oxidized, the fraction of the total tritium recovered in acetic acid after oxidation is a minimum estimate of the tritium in the p-position of alanine-t. * In each of the experiments listed in lines 3-7, samples of the same batch of alanine-t were employed as a control. Line 2 gives the mean of these separate determinations. The nL-alanine-p-t of line 1 served as a control for the experiment of line 8. c The samples obtained by HCl hydrolysis or otherwise were purified by combinations of the following: (Method a) paper electrophoresis at pH 1.9, and descending chromatography on Whatman 3 MM paper with (Method b) 1-butanol-acetic acid-water (4:1:5 by vol, upper phase) or (Method c) with 2-butanol-3y0 NH,OH (3:l by vol). Line 1 (Method b), 2(b), 3(a), 4(a), 5(a), 6(b), 7(a, b, c), 8 (b, a, c). d For the nisin experiments see Results section.

the alanine-t released by 6 E HCl for 18 hr. Most of the tritium in the residues not present in alanine-t did not migrate upon electrophoresis at pH 1.9 and was probably associated with protein or large peptide fragments. ‘The release of alanine-t by 0.03 N HCl suggests either that the alanyl-t residue in the tritiated protein is present in an unusual peptide sequence (35), or that it is linked to the rest of the protein by other t,han standard peptide bonds. Proteolysis. Undenatured Type I tritiated enzyme was very resistant to degradation by proteolyt’ic enzymes. Several attempts t’o obtain alanine-t by t,he action of trypsin and then pronase were unsuccessful. By starting with a sample of Type I tritiated enzyme containing a large amount of radioactivity (850,000 cpm) and using a very much higher ratio of trypsin and pranase to tritiated enzyme than is normally employed in degradation studies, a small amount (5000 cpm) of alanine-t was eventually isolated by electrophoresis at pH 1.9 and shown to have most of it’s tritium in the

P-position (Table III). The low yield may have occurred because a Type I to Type II transformation took place as soon as the protein began to denature, or because the tritium in the alanyl-t cont’aining pept,ides is labile. Compound B and the Formation II Tritiated Enzyme

of Type

Compound B, obtained from Type II tritiated enzyme by HCl hydrolysis, could not be extracted into ether, benzene, or ethyl acetate from neutral or acidic solutions. The compound migrated upon electrophoresis at pH 1.9 only to an extent attributable to endosmotic flow (Fig. 2) and also had zero charge at pH 5.7 and 10.3. It, therefore, lacks both carboxyl and amino groups and is not a readily hydrolyzable lactone. On thin-layer chromatography it had an RF value of 0.6 (slightly greater than that of alanine) in l-butanol-acetic acidwater (4: 1:1 by vol), 0.5 (cf alanine, 0.6) in 1-propanol-34 % NH,OH (7: 3 by vol), and like alanine did not migrate in benzene-

12

HANSON

acetic acid-water (6: 7: 3 by vol). The compound appeared to be unchanged, as indicated by electrophoresis and thinlayer chromatography, after attempted acylation with maleic anhydride in an acetone-water solution at 23”, and also after treatment with NaI04. As Compound B is obtained after 18-hr hydrolysis in 6 N HCl it is probably not a diketopiperazine. Because Type I tritiated enzyme changed to Type II in dilute solutions, it seemed likely that a partial unfolding of the protein was required before the transformation could take place. The denaturation of Type I tritiated enzyme with guanidine hydrochloride was, therefore, investigated. Brief treatment at 0” with 6 N guanidine hydrochloride followed by prolonged dialysis against water resulted in the loss to the dialyzate of 50 % of the tritium originally present. The dialyzate was sublimed, and all of the tritium in the sublimate, roughly 25 % of the original tritium, was found to be exchangeable. The tritium in the residue was probably located in Compound B, but the large amount of salt present made identification difficult. The denatured protein after being dialyzed yielded Compound B and exchangeable tritium (three samples) in roughly equal amounts upon HCI hydrolysis, i.e., the product behaved as denatured Type II tritiated enzyme. The same result was obtained if NaBH4 (5 mM final concn) was added to t,he denatured protein prior to hydrolysis. The denaturation experiment was repeated with a mixture of 6 N guanidine hydrochloride, mercaptoethanol, and iodoacetamide in the amounts required to cleave all disulfide bridges and block them against reoxidation (see Methods under Proteolysis) . Compound B and exchangeable tritium (two samples) were again obtained upon HCl hydrolysis. After treating the cleaved and blocked material with trypsin and then with pronase, Compound B was obtained (see Methods). A negligible amount of exchangeable tritium was released during the process. Tritium was released, however, when a sample of Compound B obtained by proteolysis was heated with 6 N HCl

AND

HAWK.

under the sbandard hydrolysis conditions. It is possible t’hat the tritiated compound attached to the peptide is a precursor of B and that the cleavage by pronase of an amide or ester linkage between peptide and precursor is followed by an elimination or cyclyzation reaction. In some experiments with 6 N guanidine hydrochloride the denatured tritiated protein on HCl hydrolysis gave Compound B and a mixture of products which migrated as if they were neutral amino acids at pH 1.9 (Fig. 2, peak B’) and pH 5.7, and which could be partially separated by thin-layer chromatography in 1-propanol-35 % NH,OH (7: 3 by vol). Compound B and these compounds were apparently unchanged after treatment with D- or L-amino acid oxidase. They were obtained from a batch of Type I tritiated protein which had previously given only Compound B when denatured and hydrolyzed. DISCUSSION

The principal finding of this study is that the HCl hydrolysis of samples of Lphenylalanine ammonia-lyase specifically inactivated and tritiated at the active site by the action of NaBH& (Type I tritiated enzyme) gave exchangeable tritium and nn-alanine-f. in which most, but probably not all, of the tritium was in the P-position. In interpreting this result we shall assume: inactivation and tritiation are one and the same process (see comments on Fig. 3), and one atom of borohydride derived hydrogen is incorporated per active site reduced (Table I), and that in Type I tritiated enzyme at least one half of the tritium is at the &position of an alanyl residue. It is probable that any tritium not in t#he P-position is in the cu-position of the alanyl residue, although the evidence for this is limited (Table III). As borohydride reduction implies attack on an electrophilic center, we postulate that the electrophilic center involved in catalysis is directly tritiated and is preserved as the p- or, possibly, the a-carbon, of the alanyl residue in the reduced enzyme. Two explanations for the presence of tritium in the p-position of an alanyl residue

DEHYDROALANINE

IN

PHENYLALANINE

suggest themselves. The first possibility is that NaBH& attacks an S- or O-substitut’ed cysteinyl or seryl residue, and that an SPJ~ displacement of the P-group takes place. Alkoxy substituents on boron increase the reducing power of borohydrides (36), and if such a reagent were formed at the active site by interaction with an amino acid side chain, a displacement reaction might occur. This hypothesis does not account for the apparent a-labeling of alaninet or the racemic nature of the alanine-t isolated, and does not suggest a mechanism for the enzyme’s catalytic activity. If the amino group of phenylalanine were to attack the p-carbon of the substituted cysteinyl or seryl residue and form a bond to that carbon by displacement, the result would be an alkylated amine-little better as a leaving group than the -NHs+ of phenylalanine itself.

-N-C-COY-

I

NaBHa

-N-CH-COYII

A more acceptable hypothesis (7) is that an alanyl residue is formed by the reduction of the double bond of a dehydroalanyl residue present at the active site (I + II, Y = N or 0). The reduction of dehydroalanyl residues after they have been generated in proteins by fi-elimination reactions has been reported (37, 38) although the rate of reduction may be slower than for an aldehyde group. A number of microbial peptides contain dehydroalanyl residues (39, 40) and we have investigated the NaBH& reduction of one of these, nisin (the sample of nisin was generously provided by Dr. Erhardt Gross (41, 42)). The tritiated alanine isolated from the HCI hydro1yzat.e of the peptide appeared to contain tritium on both the (Y- and P-positions although the proportions varied amongst the samples examined (Table III). At least one of the dehydroalanyl residues of nisin is readily hydrolyzed to an amide-pyruvoyl system and most isolated samples of the antibiotic exist in this form to an appreciable extent. The major product of the reduction was, therefore, tritiated lactic

AMMONIA-LYASE

13

acid. Tritiated lactic acid has not been encountered as a product of phenylalanine ammonia-lyase reduction and hydrolysis. The extent to which a dehydroalanyl residue in a protein or complex peptide can be changed to the amide-pyruvoyl form must depend greatly on the conformational flexibility of its environment. If a dehydroalanyl residue is present, it is most unlikely that it occurs in an orthodox peptide chain. Firstly, when T- adds to the P-position of the double bond and H+ to the a-position one enantiomer should be preferentially formed as the two faces of the double bond must be in very different environments and H+ cannot add to both faces of the a-carbon with equal facility. As nn-alanine-0-t is isolated, (Table 11) racemization must have taken place during hydrolysis. The racemization of amino acids upon protein hydrolysis occurs very rarely (43). The release of tritium upon hydrolysis points to the same conclusion. Tritium in the a-position would be lost upon racemizntion, and as will be indicated below, release of tritium from the p-position could also occur. Secondly, the Type I tritiated enzyme which presumably contains tritiated alanine yields nn-alanine-6-t under exceptionally mild conditions of hydrolysis. Thirdly, Type I tritiated enzyme undergoes a facile rearrangement to Type II tritiated enzyme in which some portion of the alanine residue is incorporated into Compound B. A dehydroalanyl residue that is part of a larger system may be much more reactive and show properties that differ from those expected from the study of dehydroalanine containing peptides (4446) and proteins (47). Racemization and tritium release could occur upon t,he HCl hydrolysis of a tritiated alanyl residue t,hrough the participation of the carboxyl carbonyl group or through some substituent on the nitrogen atom. If in Type I tritiated enzyme the nitrogen of the alanyl-t residue is acylated and the other point of attachment (Y) is a good leaving group, then on acid hydrolysis the transient formation of a saturated azlactone could account for both racemization and loss of cY-tritium as H-C-C=0 ti

14

HANSON

C”=C-OH tautomerism would take place (48, 49). The recent work of Givot et al. (9) on histidine ammonia-lyase, however, suggests that the N-substituent is responsible. By studying the products obtained from proteolysis, and reduction of nitromethane-14C-inactivated enzyme they concluded that a Schiff’s base of a dehydroalanyl residue is present at the active site (III). If Type I tritiated enzyme contains a Schiff’s base of an alanyl residue (IV), then tautomerism of the type C=N-CPH s HC-N--“C would account for racemization and loss of any cr-tritium. Tritium could also be lost from the P-position through further tautomerism and by way of hydrolysis to give pyruvate-3-t.

AND

HAVIR

conjugated system of the prosthetic group of both enzymes. Mechanism

Any proposal concerning t’he prosthetic group of an enzyme must be able to account both for the chemical evidence and for the enzyme’s catalytic properties. The above results indicate that the @position of a dehydroalanyl residue is the electrophilic center of the active site. For dehydroalanyl residues in peptides this is the favored position for attack by such soft nucleophils (50) as thiols and amines (4446). Crossconjugation of t’he 01,&double bond to a Schiff’s base would be expected to reinforce the tendency to p-addition and to facilitate elimination from t’he adduct Indeed, the enzymatic replacement of one fi-substituent NaBH4 of alanine by another, e.g., OH, SH, and -C=N-C-COYindole, is believed to take place through the CJ& conversion of their pyridoxal 5-phosphate I I Schiff’s base derivatives to a Schiff’s base -C==N-CH-COYof dehydroalanine (51, 52a). In Fig. 5 such IV III a p-addition is shown as Step 2.1. Some inductive withdrawal of electrons from the There is every reason to believe that the nitrogen of the phenylalanine must occur prosthetic groups of phenylalanine and in the product of this reaction (bl) but it histidine ammonia-lyase are identical. Winkseems unlikely’ that t,his would constitute ler (10) has recently shown that histidine the good leaving group required for the ammonia lyase inactivated and tritiated elimination process. As the hard nucleowith NaBH4-t4 yields alanine-t upon HCl phils OH and HZ0 add to the cu-position of hydrolysis and, although we have only dehydroalanyl residues we have considered carried out preliminary studies on the nitrothe possibility that the nitrogen of phenylmethane inactivation of phenylalanine am- alanine adds to the a-posit’ion (8). The monia-lyase, we have established that more intermediate would t,hen be analogous to than 1 mole of nitromethane-14C is incor- the carbinolamine previously thought to porated per mole of enzyme and that nitrobe an intermediate on the basis of the carmethane-inactivated enzyme is not tribonyl-like characteristics of the prosthetic tiated by NaBH4-t4. (We are indebted to groups of histidine (5) and phenylalanine Drs. Abeles and Givot for making their ammonia-lyases (3, 6). Such an intermeresults available to us prior to publication diat’e would likewise function through the (9).) It must be presumed that, as in the inductive withdrawal of electrons. case of histidine ammonia-lyase, nitroNeither of the above alternatives has methane adds to the /Lposition of the double seemed to us to be very satisfactory. One bond and that the imino group is inacces- would prefer to be able to write a mechasible to attack by such reagents as boro- nism in which the free energy of the transihydride) nitromethane, cyanide, etc. As tion state for the bond-breaking process neither enzyme contains pyridoxal phos- is lowered by resonance. This condition phate or shows any measurable spectral can be satisfied if the intermediate bl changes upon reduction, it seemsprobable undergoes a rearrangement. The iminethat the partial formula III defines the enamine transformation shown (Step 2.2) f

I

DEHYDROALANINE CATALYTIC

IN

PHENYLALANINE

15

CYCLE

I.-?-ADDITION

,3

STEPS

I

AMMONIA-LYASE

- PROTOTROPK

SHIFT

e ELIMINATION

STEPS

3 STEP

FIG. 5. P-Addition mechanism. The nitrogen of the dehydroalanyl residue is present as a Schiff’s base inaccessible to nucleophilic attack by free reagents; Y is either N or 0; R is either H or the rest of the phenylalanine molecule. The configurations and relative orientations of the double bonds are chosen to permit hydrogen bonding analogous to that held to occur in the tautomeric form of pyridoxal phosphate Schiff’s bases (52b) and to give maximum stability to the system C=N-C”=C@. A suprufacial migration (53 cf. 54) is indicated for the 1,3-prototropic shift on the assumption that the rearrangement is catalyzed by the group B which must be on the side of the plane of the prosthetic group opposite to the substrate amino group. The resonance stabilization of the transition state for elimination (Step 3) is indicated by the partial formula in curved brackets. The carbonyl group may well be protonated rather than charged in this state. The stereochemistry for the substrate shown is based on experiments with (flR)-Lphenylalanine-p-t obtained by rational synthesis (8) and corresponds to the stereochemistry recently established for histidine ammonia-lyase (9, 55); the pro-S hydrogen is eliminated.

maintains conjugation to the C=O group and is analogous to the prototropic shifts believed to occur in a variety of enzymecatalyzed aldol condensations (52b). The product (b2) may be regarded as the vinylogue of an amide, i.e., O=C-0=(3--N compared to O=C-N. More precisely, the leaving group is a vinylogue of a carbamyl ester or of urea, depending on the

nature of Y. An enzymatic analogy for the elimination step exists in the conversion of the amide a-AICAR-succinate to AICAR and fumarate by adenylosuccinate ,4MPlyase (56). Chemically the elimination could be written as a stepwise or a concerted process; however, if the elimination is concerted it may be regarded as t,he acyclic equivalent of the pyrolgtic &elimination

16

HANSON

of amides-a process involving a cyclic transition state stabilized by resonance (57). After cinnamate loss (Step 4), a return prototropic shift (Step 5.1) and 1,4elimination (Step 5.2) leads to free NH,+ (Step 6). Insofar as the above mechanism accounts for the enzyme’s function it supports the view that the prosthetic group contains a Schiff’s base of a dehydroalanyl residue. If the structure of the protein is necessary for the integrity of the prosthetic group, as it is for the retention of the Type I form of the reduced enzyme, progress in determining its full structure will continue to require the isolation and characterization of fragments derived from the active site. ACKNOWLEDGMENT We express our appreciation to Katherine Clark and George R. Smith for their skillful nical assistance.

A. tech-

REFERENCES 1. KOUKOL, J., AND CONN, E. E., J. Biol. Chem. 236, 2692 (1961). 2. HAVIR, E. A., AND HANSON, K. R., Biochemistry 7, 1896 (1968). 3. HAVIR, E. A., AND HANSON, K. R., Biochemistry 7, 1904 (1968). 4. HODGINS, D. S., Biochem. Biophys. Res. Commm. 32, 246 (1968). 5. SMITH, T. A., CORDELLE, F. H., AND ABELES, R. H., Arch. Biochem. Biophys. 120, 724 (1967) . 6. PETERKOFSKY, A., J. Biol. Chem. 237, 787 (1962) . 7. HANSON, K. R., AND HAVIR, E. A., Fed. Proc. 26, 602 (1969). 8. HANSON, K. R., AND HAVIR, E. A., in “Recent Advances in Phytochemistry, 4” (J. Watkins and V. C. Runeckles, eds.) (Proc. Symp. Phytochem. Sot. N. Amer., Aug. 1969), in press. Appleton-Century-Crofts, New York. 9. GIVOT, I. L., SMITH, T. A., AND ABELES, R. H., J. Biol. Chem. 244, 6341 (1969). 10. WINKLER, R. B., J. Biol. Chem. 244, 6550 (1969). 11. HAVIR, E. A., AND HANSON, K. R., Methods Enzymol. 17a, 575 (1970). 12. MARSH, H. V., JR., HAVIR, E. A., AND HANSON, K. R., Biochemistry 7, 1915 (1968). 13. HAVIR, E. A., REID, P. D., AND MARSH, H. V., JR. In preparation.

AND

HAVIR

14. MOORE, S., AND STEIN, W. H., Methods Enzymol. 6,819 (1963). 15. ENGLARD, S., AND H.~NSON, K. R., Methods Enzymol. 13, 567 (1969). 16. MOORE, S., J. Biol. Chem. 243, 6281 (1968). 17. CALKINS, V. P., Anal. Chem. 16, 762, (1943). 18. ZELITCH, I., J. BioZ. Chem. 233, 1299 (1958). 19. ZELITCH, I., J. Biol. Chem. 240, 1869 (1965). 20. DENISON, F. W., JR., AND PHARES, E. F., Anal. Chem. 24, 1628 (1952). 21. GILVARG, C., AND BLOCH, K., J. Biol. Chem. 193, 339 (1951). 22. JOHNS, R. B., AND WHELAN, D. J., Aust. J. Chem. 19, 2143 (1966). 23. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J., J. Biol. Chem. 193, 265 (1951). 24. DELANGE, R. J., GLAZER, A. N., AND SMITH, E. L., J. Biol. Chem. 244, 1385 (1969). 25. BERGMANN, M., AND KBSTER, H., 2. Physiol. Chem. 169, 179 (1926). L., OT~NI, T. T., WINITZ, M., AND 26. ZERVSS, GREENSTEIN, J. P., J. Amer. Chem. Sot. 61, 2878 (1959). 27. BERGMANN, M., AND ZERVAS, L., 2. Physiol. Chem. 162, 282 (1926). M., NEIDERWIESER, A., AND 28. BRENNER, PATAKI, G., in “Thin Layer Chromatography” (,E. Stahl, ed.) p. 400. Academic Press, New York (1965). 29. KLEBE, J. F., FINKBEINER, H., AND WHITE, D. M., J. Amer. Chem. Sot. 88, 3390 (1966). 30. STALLING, D. L., AND GEHRKE, C. W., Biothem. Biophys. Res. Commun. 22, 329 (1966). 31. LAMKIN, W. M., AND GEHRKE, C. W., Anal. Chem. 37, 383 (1965). 32. GROS, C., AND LABOUESSE, B., Eur. J. Biothem. 7, 463 (1969). 33. GRAY, W. R., Methods Enzymol. 11, 139 (1967). T., AND TAMIY.~, N., Biochem. J. 78, 34. OSHIMA, 116 (1961). 35. TSUNG, C. M., FR~ENKEL-CONRAT, H., Biothem. 4, 793 (1965). 36. BROWN, H. C., “Hydroboration,” p. 248. Benjamin, New York (1962). 37. BAYER, E., AND PARR, W., Angew. Chem. Int. Ed. Engl. 6, 840 (1966). 38. TANAKA, K., BERTOLINI, M., AND PIGMAN, W., Biochem. Biophys. Res. Commun. 16, 404 (1964). B. W., Nature London 224, 696 39. BYCROFT, (1969). 40. GROSS, E., MORELL, J. L., AND CRAIG, L. C., Proc. Nat. Acad. Sci. U.S.A. 62, 952 (1969). 41. GROSS, E., AND MORELL, J. L., J. Amer. Chem. Sot. 89, 11 (1967). 42. GROSS, E., AND MORELL, J. L., Fed. Eur. Biol. Sot. Letters 2, 61 (1968).

DEHYDROALANINE

IN

PHENYLALANINE

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