THE JOURNAL OF B~omorca~ CHEMISTRY Vol. 243, No. 1, Issue of January 10, pp. 178-185, 1968

Printed in U.S.A.

Threonine Deaminase from Salmonella typhimurium

I. PURIFCATION AND PROPERTIES*

(Received for publication, July 27, 1967)

R. 0. BURNS AND MARIO H. ZARLENGO

From the Department of Microbiology and Immunology, Duke University Xchool of Medicine, Durham, North Carolina 27706

SUMMARY

Biosynthetic L-threonine deaminase (L-threonine hydro- Iyase, deaminating; EC 4.2.1.16) has been purified approxl- mately 250-fold from crude extracts of nutritionally de- repressed Salmonella typhimurium. Sedimentation of the enzyme in the ultracentrifuge results in a single, sharp, symmetrical peak in the schlieren pattern. The purification procedure is capable of removing the radioactivity from a crude extract containing label in all the proteins except native threonine deaminase.

The deaminase reaction shows homotropic interaction only in the presence of L-isoleucine; high ionic strength or low valine concentrations remove these interactions. The pure enzyme is effectively inhibited by L-isoleucine. The pH optimum of the deaminase reaction is 9 to 10; the pH curve is shifted to higher values in the presence of L-isoleucine. The activity of the purified enzyme is independent of added pyridoxal phosphate. The cofactor is resolved by dialysis against tris(hydroxymethyl)aminomethane hydrochloride buffer at pH 8.0. Pyridoxal phosphate and pyridoxamine phosphate are capable of reactivating the enzyme. The activity of the purified enzyme is independent of divalent cations, but NH*+, Kf, Lif, and Na+ show about 20% stimulation of enzyme activity.

The enzyme has two absorption maxima (280 and 420 mp). The removal of pyridoxal phosphate from the enzyme is accompanied by the loss of the 420 rnp absorption. The enzyme contains 0.99 mole of pyridoxal phosphate per 100,000 g of protein. The minimum molecular weight computed on the basis of pyridoxal phosphate content and sedimentation coefficient is 200,000.

The modification of the activity of certain enzymes by specific metabolites is recognized as a means whereby the proper func- tion and integration of metabolic pathways is maintained, and

* This work was supported by Grant GM-12551 from the Na- tional Institutes of Health.

the significance of these regulatory enzymes for ensuring cellular economy is well known (1). It has, for example, become a biochemical precept in microbiology that the end product of a biosynthetic pathway ought to exert a negative feedback control on the activity of at least one enzyme in the pathway (2).

The activity of enzymes which are controlled by this negative feedback inhibition is frequently stimulated by still other metabolites. In both instances, the structures of these positive and negative effecters of enzyme activity bear little resemblance to the structure of the substrate (or substrates) for the pertinent enzyme (3, 4). Relatively little is known about the structure of these regulatory enzymes, and the current views of how small molecules cause changes in the catalytic properties come pri- marily from indirect lines of evidence. Kinetic analysis of a number of regulatory enzymes has led to the speculation that these catalysts contain multiple active centers and therefore possess a distinct quaternary structure composed of multiple subunits. These observations, plus the demonstration that various modes of treatment are able to separate the catalytic activity of an enzyme from its ability to be inhibited by the pertinent metabolite, have led to the well known allosteric model (5, 6). The essential features of this model state that an allosteric effector, binding at a site on the enzyme molecule distinct from the active site, induces a conformational change in the enzyme so as to increase the association between subunits, and thus inhibit enzyme activity, or, conversely, to relax this association and increase enzyme activity.

L-Threonine deaminase (L-threonine hydro-lyase, deaminat- ing; EC 4.2.1.16) is the initial enzyme in the pathway of L-

isoleucine biosynthesis in Salmonella typhimurium as well as in a variety of other microorganisms. This enzyme, besides being the first to be described prominently with respect to the phys- iological function of end product inhibition (7), has been the subject of much recent work on which our current notions of regulatory enzymes are based. The activity of L-threonine deaminase is inhibited by L-isoleucine and stimulated under appropriate conditions by low concentrations of L-valine (8, 9). The physiological significance of the former observation is obvious; the significance, if any, of the latter observation is not so clear. The present study was undertaken in an effort to elucidate the structural characteristics of L-threonine deaminase as related to its regulatory properties.

178

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Issue of January 10, 1968 R. 0. Burns and M. H. Zarlengo 179

EXPERIMENTAL PROCEDURE

Cultivaticnz of Organisms and Preparation of Extracts-Cells enriched for threonine deaminase were obtained as follows. Strain leu A-124, a stable leucine auxotroph derived from Salmonella typhimurium strain LT-2, was grown with aeration in la-liter quantities of Davis-Mingioli medium (10) modified by omitting the citrate and increasing the glucose to 0.5%. This medium was supplemented with 50 mg of L-isoleucine and 5 mg of L-leucine per liter. This concentration of leucine is growth- limiting. Upon termination of growth at 37”, 1 liter of a 0.024% solution of L-leucine was pumped into the culture medium over a period of 6 hours; this procedure permitted the culture to undergo two more generations at a growth rate limited by the availability of leucine. By taking advantage of the principles of multi- valent repression (11) in this manner, a derepressed rate of synthesis of threonine deaminase was achieved. The isoleucine in the medium competes with leucine for uptake by the cells and ensures limiting conditions with respect to leucine (12, 13). The cells were harvested by means of a Sharples centrifuge.

Threonine deaminase containing tritium-labeled pyridoxal phosphate was obtained from cells cultured as described above, but in a medium supplemented with 0.01 mg of pyridoxine per liter, with a radioactivity of 6 PC per pmole. In this case the organism used was S. typhimurium strain leu A-124 with a requirement for vitamin Bs; this strain was obtained by trans- ducing the Zeu A-l.24 locus into a B6 strain (obtained from Dr. Martin Freudlich). The transducing phage was PLT 22, and standard techniques were used. The Bc- Zeu A-124 strain was freed of phage before use. The use of a Bg auxotroph prevented dilution of the exogenously supplied vitamin by endogenously formed cofactor.

Protein labeled with s5S was prepared from S. typhimurium strain leu A-124 grown in a medium containing the following, per liter: K2HPO,, 7.0 g; KH2P0,, 3.0 g; MgSOa, 0.1 g; NH&l, 1.0 g; and 1.0 mC of 35S as NazS04. A chemostat was used to obtain cells with maximally derepressed levels of threonine deaminase.

Enzyme Assays-Threonine deamination was followed by measuring the Lu-ketobutyrate formed as the phenylhydrazone derivative, as described by Umbarger and Brown (14). The reaction mixture contained, per ml, Tris-HCl, pH 8.0, 106 pmoles; NH&l, 100 pmoles; and L-threonine, 80 pmoles. The reaction was started by the addition of enzyme and terminated by trans- ferring an appropriate portion of the reaction mixture directly to 3 ml of the 2,4-dinitrophenylhydrazine reagent. Assays were performed at 34”. A unit of threonine deaminase activity is defined as the amount of enzyme forming 1 pmole of a-keto- butyrate per hour. The specific activity is the number of units of enzyme per mg of protein. Rate studies with the purified enzyme were performed by the use of a modification of thecoupled assay of Maeba and Sanwal (15). The basic reaction mixture contained: Tris-HCl, pH 8.0, 100 pmoles; bovine heart lactic dehydrogenase, 0.8 mg; and NADH, 0.3 pmole. The total reaction volume was 3.0 ml in a standard l-cm cuvette, and the temperature was 2425”. The amount of lactic dehydrogenase varied, depending upon the activity of the particular preparation of this enzyme used. Preliminary assays were performed in order to determine conditions for first order reaction rates with respect to substrate. The velocity of the reaction is defined as the change in absorbance per min. Other additions to the reaction mixture are described below. A Gilford model 1000

multiple sample absorbance recorder was used to monitor the disappearance of NADH.

Measurement of Pyridoxal Phosphate-Pyridoxal phosphate was assayed with apotryptophanase. This enzyme was prepared from Escherichia coli by the procedure of Burns and DeMoss (16). The tryptophan was essentially free of pyridoxal phos- phate, and a double reciprocal plot of the concentration of pyridoxal phosphate with respect to the initial velocity was used as a standard curve for the cofactor. Several dilutions of the solutions to be measured were assayed to ensure validity of the standard curve. The tryptophanase reaction, which yields indole, pyruvate, and ammonia, was coupled with an excess of lactic dehydrogenase, and the oxidation of NADH was followed spectrophotometrically. The K, of tryptophanase for pyr- idoxal phosphate is about 1.0 X 10-C M; this system therefore provides a sensitive assay for pyridoxal phosphate. The details of this procedure will be published elsewhere. Two other methods were also used for measuring pyridoxal phosphate; these are described under “Results.”

Protein Measurement-Concentration of protein was deter- mined by the method of Lowry et al. (17) and by the ratio of absorbances at 280 and 260 rnp (18).

Physical Techniques-Sedimentation velocity experiments were performed in a Spinco model E centrifuge equipped with schlieren optics and an RTIC temperature control unit. Tritium was measured in a Packard Tri-Carb liquid scintillation counter. Radioactive sulfur was measured in a Nuclear-Chicago model 186 gas flow counter. Spectral analyses were performed in a Cary model 15 spectrophotometer.

Chemicals-Amino acids, dithiothreitol (Cleland’s reagent), pyridoxal phosphate, and pyridoxamine phosphate were obtained from Calbiochem. NADH was a product of P-L Biochemicals. Tritiated pyridoxine was purchased from Tracerlab, and Na#%Oa was obtained from New England Nuclear. Lactic dehydrogenase was obtained from Worthington. DEAE-cellulose was a product of the Brown Company. Brushite was prepared ac- cording to the method of Tiselius, Hjerten, and Levin (19). All other chemicals were reagent grade.

RESULTS

Enzyme Puri$cation

Preparation of Crude Extract-Cells obtained from 96 liters of culture medium were washed once with 0.05 M potassium phos- phate, pH 7.4, containing 8 X 10-d M L-isoleucine, 5 X 10m4 M

EDTA, and 5 x low4 M Cleland’s reagent. The cells were re- suspended in 200 ml of the same buffer and disrupted in the cold by use of a IlO-watt Branson Sonilier at full power for 10 min. The crude extract was cleared of debris by centrifugation at 10,000 x g for 20 mm. The debris and remaining intact cells were resuspended in the same buffer and were again subjected to sonic vibration for 10 min. The supernatant liquids were combined and immediately used for the purification of threonine deaminase.

The supplements added to the buffer aided in stabilizing the threonine deaminase. It is well known that L-isoleucine, an inhibitor of the activity of this enzyme, is particularly useful for this purpose (20), and the chelating agent and dithiol augmented this stabilization; other thiols such as P-mercaptoethanol or glutathione would not replace Cleland’s reagent.

Diethylaminoethyl Cellulose Chromatography-The chromatog-

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180 Threonine Deaminase from S. typhimurium. I Vol. 243, No. 1

raphy, as well as all subsequent purification steps, was carried out at 4’. The crude extract was divided into two equal por- tions. Each portion was placed directly on a DEAE-cellulose column (5 x 35 cm) which had been previously washed with KC1 (1 M) and equilibrated with 0.02 M potassium phosphate, pH 7.4, containing 8 X 10T4 M n-isoleucine, 5 X 10e4 M EDTA, and 5 x 10m4 M Cleland’s reagent. The protein was eluted with a linear gradient consisting of 2 liters of 0.02 M potassium phos- phate, pH 7.4, containing 8 X low4 M n-isoleucine, 5 X lo-4 M EDTA, and 5 X 1Oe4 M Cleland’s reagent in the mixmg chamber, and 2 liters of the same buffer made 0.4 M with respect to KC1 in the reservoir. The threonine deaminase was eluted at about 0.35 M KCl. Fractions containing the enzyme at a specific activity of at least 1200 were pooled and retained for further purification.

The enzyme solution at this stage is fairly dilute and contains high concentrations of KCl; these are severe conditions with re- spect to enzyme stability. Therefore, the enzyme preparation was immediately carried through the next step of the purification procedure.

Chromatography on Calcium Phosphate-This step, besides re- sulting in a purification, removes the KC1 and concentrates the enzyme. The calcium phosphate used was brushite. This form of the absorbent possesses a lower capacity for the enzyme than does hydroxylapatite, and much larger columns must be used. Nevertheless, ease in preparation and more desirable elution rates made brushite the adsorbent of choice for this particular pro- cedure. The pooled fractions from each DEAE-cellulose col- umn were placed on brushite columns (5 X 35 cm) which had been equilibrated with 0.02 M potassium phosphate, pH 7.6, supplemented with 6 X 10m4 M n-isoleucine, 5 X 10-h M EDTA, and 5 x 10m4 M Cleland’s reagent. The enzyme was eluted with a linear gradient consisting of 2 liters of 0.02 M potassium phos- phate, pH 7.6, in the mixing chamber and 2 liters of the same buffer at 0.05 M in the reservoir. Both of these buffers were supplemented with 6 X 10e4 M n-isoleucine, 5 X 10m4 M EDTA, and 5 X 10m4 M Cleland’s reagent. The enzyme was eluted after

TABLE I Purijkalion of threonine deaminase

Fraction Total protein

,,ig Crude extract. ............. 8,700 DEAE-cellulose ........... 481 Calcium phosphate. ...... 54 (NH4)zSOa (40-55s) ...... 29 Sephadex G-200 ........... 11

wails mits/mg 948,960 113 591,000 1,230 389,736 7,200 380,000 14,000 316,180 27,000

Enzyme dis- carded after

each step

155,400 76,752

27,080

FIG. 1. Schlieren patterns of purified threonine deaminase sedimenting from left to right. Photographs were taken at 4-min intervals after attaining a rotor speed of 59,780 rpm. Protein, 0.59% in 0.1 M potassium phosphate, pH 7.0. A double sector cell was used, and the temperature was 25”.

approximately 750 ml of the gradient had passed through the column and was usually contained in about 150 to 200 ml. The fractions from both columns containing the enzyme at a specific activity of at least 6,000 were pooled and concentrated by vac- uum dialysis; the dialysis bag was partially covered with 0.05 M potassium phosphate, pH 7.4, containing 8 X 10m4 M n-isoleucine, 5 X 10-d M EDTA, and 5 X 10e4 M Cleland’s reagent. Fre- quently, the enzyme obtained from this procedure had a specific activity of 12,000 or greater. If so, the ammonium sulfate step described below was omitted and the subsequent gel filtration was performed.

Ammonium Sulfate Precipitation-The concentrated prepara- tion, containing about 5 mg of protein per ml, was made 40% saturated with respect to ammonium sulfate, and the slight precipitate was centrifuged and discarded. The supernatant was then made 55% saturated with ammonium sulfate. The precipitate was centrifuged and redissolved in a minimum of 0.05 M potassium phosphate, pH 7.4, containing 8 X 10e4 M r;-isoleucine, 5 X lo+ M EDTA, and 5 X 10v4 M Cleland’s re- agent. The recovery of the enzyme from this step was variable; frequently as much as 90 to loo%, but at other times only 60% of the enzyme was recovered.

During preliminary attempts to purify this enzyme by am- monium sulfate precipitation of crude extracts, it was found that considerable inactivation occurred. It is clear from the above observations that partially purified and concentrated enzyme is much more refractory to inactivation by this treatment.

Sephudez G-200 Filtration-The ammonium sulfate fraction, or the concentrated high activity fraction from the brushite treatment, was passed through a column (2 x 150 cm) of Sephadex G-200 equilibrated with 0.05 M potassium phosphate, pH 7.4, containing 8 X 10e4 M n-isoleucine, 5 X 10e4 M EDTA, and 5 X lo-4 M Cleland’s reagent. The enzyme was eluted with the same buffer and appeared slightly behind the front. The en- zyme was concentrated as described above, dialyzed against 0.05 M potassium phosphate, pH 7.4, containing 8 X 1W4 M n-isoleucine, 5 X 10d4 EDTA, and 5 X 10m4 M Cleland’s reagent, and stored at -20”. The concentrated enzyme was stable if stored frozen (-20”) in 0.05 M potassium phosphate, pH 7.4, containing 8 X 10e4 M n-isoleucine, 5 X 10m4 M EDTA, and 5 X 10s4 M Cleland’s reagent. No loss of activity was detected after several weeks. Table I presents a summary of the results of the purification procedure. The total recovery from each step of the purification is given by the sum of the units in the columns headed “Enzyme retained . .” and “Enzyme dis- carded. . . .”

Purity of Enzyme

The final enzyme preparation was examined with respect to its sedimentation properties. Fig. 1 is a series of photographs rep- resenting the boundary of the enzyme during a sedimentation analysis. The sz~+ extrapolated to zero protein concentration was 8.7 (21). The purified protein sedimented as a single com- ponent with a symmetrical schlieren peak at all concentrations tested.

Although the purification procedure resulted in a good re- covery of the initial enzyme activity, there was a significant in- activation of the enzyme. It will subsequently become evident that it is necessary to know whether the enzyme preparation ob- tained by the purification procedure contains active as well as inactive enzyme protein; that is, whether or not the inactive en-

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zyme copurifies with the active enzyme. The following experi- ment answers this question and provides additional information on the purity of the preparations. A small amount of radio- active, crude extract was prepared from S. typhimurium. This was accomplished by growing the organism in the presence of 35S (see “Experimental Procedure”). The conditions of cultiva- tion (a chemostat) were such as to ensure a high rate of produc- tion of threonine deaminase, and this was confirmed by extracting a small portion of the cells in 0.05 M potassium phosphate, pH 7.4, containing 8 X 10m4 M n-isoleucine, 5 X low4 M EDTA, and 5 X 10m4 M Cleland’s reagent, and measuring the enzyme. The remainder of the cells were extracted in 0.05 M potassium phos- phate, pH 7.4, containing 5 x 10m4 M EDTA and 5 x 10m4 M Cleland’s reagent (the isoleucine was omitted). The extract was dialyzed overnight against 500 volumes of 0.05 M potassium phosphate, pH 7.4, and then placed at 37” in order to inactivate the threonine deaminase. When no detectable activity re- mained, the radioactive extract was added to a standard prepara- tion of crude extract containing active threonine deaminase. This procedure effectively labels all of the protein of the prepara- tion, with the exception of native threonine deaminase (it should be noted, however, that labeled inactivated enzyme is still pres- ent). The mixture of crude extracts was then carried through the purification procedure. Fig. 2 shows the elution pattern of protein, enzyme, and radioactivity from the final step of the purification, the Sephadex G-200 column. The profile shows that the first protein peak and the enzyme activity coincide, and the radioactivity is confined to the second peak.

It should be pointed out here that threonine deaminase does, in fact, contain cysteine and methionine (21). These results in- dicate that the inactive threonine deaminase is removed during the course of purification, and, although it is not evident at which step this removal occurs, the results increase the probability that the final preparation is void of inactive enzyme. The radio- activity of the mixture of crude extracts was 4500 cpm per mg of protein, whereas the radioactivity of the pooled fractions from the Sephadex column was 500 cpm per mg of protein, indicating that the threonine deaminase was about 90% pure. The data presented in the accompanying paper (21) also provide evidence for the homogeneity of the enzyme preparation.

Allosteric Properties of Purified Enzyme

The present investigation was undertaken in an effort to corre- late the structure of the enzyme with its functional character- istics. The activity of this enzyme is effectively inhibited by n-isoleucine and is stimulated under appropriate conditions by L- valine (8, 9). The capacity of an enzyme to exhibit these char- acteristics can usually be separated from the catalytic properties. The biosynthetic threonine deaminases from Escherichia coli (9, 20), S. typhimurium (15), and Rho&pseudomonas spheraides (22) are subject to modification with respect to inhibition by L- isoleucine; in all cases, mercurials are able to dissociate the cata- lytic from the inhibitory property. Manipulation of the enzyme during the course of purification could have resulted in spontane- ous loss of the feedback site on the enzyme, but if the material ob- tained from the purification procedure is to be suitable for further analysis it must exhibit these native properties. Fig. 3A shows that the curve relating initial velocity of the enzymatic conver- sion to substrate concentration is a rectangular hyperbola. Ad- dition of low levels of isoleucine to the assay mixture results in a sigmoidal relationship of the reaction velocity to substrate con-

FIG. 2. The separation of radioactive protein from native threo- nine deaminase; elution patterns from Sephadex G-200 columns. The conditions of the filtration are presented in the text.

centration. In addition, if the isoleucine is present together with relatively high levels of KC1 (Fig. 3B) or low levels of valine (Fig. 3C), typical Michaelis-Menten kinetics results. These re- sults are readily interpreted in terms of the allosteric model presented by Monod, Wyman, and Changeux (6). The enzyme exists largely in the “relaxed” form, and the “relaxed-tight” reaction (R + T) is shifted, by the binding of isoleucine, to the right, thereby bringing about an interaction between active sites on the enzyme molecule. Removal of this hypothetical interaction by the KC1 could be attributed to a nonspecific inter- ference with the forces involved in the quaternary structure of the enzyme, whereas perturbation of the same interaction by valine might be visualized as a consequence of the stereospecific binding of this amino acid to the enzyme. These results are in contra- diction to those previously reported from this laboratory. In a preliminary report (23) it was stated that the S. typhimurium threonine deaminase activity exhibited sigmoidal kinetics at low ionic strength and Michaelis-Menten kinetics at high ionic strength. These observations can now be explained by the presence of low levels of isoleucine carried over from the enzyme preparation to the previous assay mixtures (the buffer in which the enzyme was dissolved contained isoleucine). The results shown here are qualitatively similar to those reported by Maeba and Sanwal (15), except that relatively lower levels of isoleucine are required in our preparations to obtain the presumed inter- action of the active sites on the enzyme molecule. The necessity for a higher concentration of isoleucine reported by Maeba and Sanwal can be attributed to the fact that their assays were per- formed in a buffer of higher ionic strength that that used here.

Fig. 4 illustrates the effect of n-isoleucine on the activity of the purified enzyme and shows that the activity of the enzyme is effectively inhibited by L-isoleucine. This observation, together with the foregoing results, indicates that the activity of the purified enzyme is sensitive to allosteric effecters and that the properties (15) of the enzyme activity in crude preparations are possessed by the purified enzyme.

Effect of pH

Fig. 5 shows the effect of hydrogen ion concentration on the rate of the deaminase reaction. The optimal activity was at pH 9.0 to 10.0. The optimal pH was shifted to higher values in the presence of the inhibitor, n-isoleucine, because the enzyme was relatively insensitive to inhibition by isoleucine at higher pH values. The results in Fig. 6 show the effect of lowering the pH of a reaction proceeding at pH 10.0. These results illustrate that the effect of isoleucine was manifest even though the enzyme had

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Threonine Deaminase from S. typhimurium. I Vol. 243, No. 1

I/l-tkreonine CiirnJ)

z =

x .6

8 .4

i.2

0 20 40 L-threonine(M)x104

01 I I I 1 3

‘IL-thfeonine (l/mn)

01 I I I I 1

l/L-th2reonine ~l?ntvl~

4

-

FIG. 3. The reaction velocity of purified threonine deaminase. The enzyme was dialyzed overnight against 0.05 M potassium phosphate, pH 7.4, plus 5 X 10m4 M EDTA and Cleland’s reagent. The coupled assay was used. A: A, no addition; 0, RF M L- isoleucine added to assay. B: 10-C M L-isoleucine added to assay, with (A) and without (0) 0.1 M KCl. C: 10-6~ L-isoleucine added to assay, with (A) and without (0) lo-” M L-valine.

been at pH 10.0, and show that the enzyme was not irreversibly desensitized by previous exposure (in the reaction mixture) to the high pH. The change in the rate of the noninhibited reaction following the change in pH simply reflects the intrinsically lower activity of the enzyme at pH 8.0.

The pH dependence of activity suggests the titration of an essential basic group in the enzyme molecule, the nonprotonated form of which would be necessary for enzyme activity. The displacement of the curve toward a higher pH range by isoleucine might then be the result of a conformational change which alters the environment of the pertinent basic group in such a way as. to bring about a shift in the pK of this group. Although this ex- planation would be consistent with the current notions of “allo- steric transitions,” there is at present no evidence to support it further. Since other explanations are equally plausible, this point is presently being investigated.

Cofactor Requirement

A spectrophotometric analysis of the purified enzyme is pre- sented in Fig. 7. The enzyme was found to have two absorption maxima, the usual one at 278 rnp and another at 410 to 420 mp. The extinction of the enzyme at 278 rnp was determined by

. 0 I I I I I

I 2 3 4 5 L-isoleucine (M_) x IO4

FIG. 4. The inhibition of purified threonine deaminase by L- isoleucine. The coupled assay was used; isoleucine was present at the concentrations indicated. The enzyme was dialyzed over- night against 0.05 M potassium phosphate, pH 7.4, with 5 X 10m4 M EDTA and Cleland’s reagent.

FIG. 5. The effect of pH on the threonine deaminase reaction. The enzyme was dialyzed overnight against 0.05 M potassium phosphate, pH 7.4, with 5 X lO+ M EDTA and Cleland’s reagent. The calorimetric assay was used, and the total assay mixture was adjusted to the desired pH with HCl. 0, without isoleucine; n 10-3 M L-isoleucine. teL) .

SA, specific activity (units per mg of pro-

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Issue of January 10, 1968 R. 0. Burns and M. H. Zarlengo 183

weighing a desiccated portion of the purified enzyme on a Cahn microbalance. A 0.1% solution of the enzyme in 0.05 M po-

tassium phosphate, pH 7.4, containing 8 X 10e4 M L-isoleucine, 5 x 10m4 M EDTA, and 5 X 10m4 M Cleland’s reagent in a stand- ard l-cm cuvette, gave an absorbance of 0.93. The value was used for quantitative measurement of pure threonine deaminase. The 410 to 420 rnp absorption is characteristic of enzymes con- taining pyridoxal phosphate as a hydrogen-bonded Schiff base, and suggests that the cofactor is associated with the enzyme by way of an azomethine bond, probably involving the t-amino

FIG. 6. The preservation of sensitivity to isoleucine during assay at high pH. A reaction was allowed to proceed at pH 10; the arrow represents the time at which samples were removed from the reaction mixture and added to reaction mixture at pH 8, with and without 10-z M n-isoleucine. The data are corrected for a lo-fold difference in activity following the dilution.

.I

1

350 400 wavelength. nw

FIG. 7. The absorption spectrum of holo- (-) and apo- (- - -) threonine deaminase. The apothreonine deaminase was obtained as described in the text. The pyridoxal phosphate-resolved en- zyme was dialyzed against 0.05 M potassium phosphate, pH 7.4, with 5 X 10-” M EDTA and Cleland’s reagent; the holoenzyme was in the same buffer. A l-cm light path was used, and the absorb- antes were normalized at 280 rnp.

TABLE II

Effect of Bg analogues on activity of apothreonine deaminase

Purified enzyme was dialyzed for 20 hours against 2000 volumes of 0.05 M Tris-HCl, pH 8.0, supplemented with 5 X 10m4 M EDTA and 5 X 1OF M Cleland’s reagent. The calorimetric assay was performed with 1 rmole of the compounds shown. Keto acid was measured at 3-min intervals for 24 min, and the maximal linear rate was taken as the reaction velocity.

Addition Specific activity

units/mg

None........................... 0 Pyridoxal phosphate. 10,800 Pyridoxamine phosphate 10,200 Pyridoxal. . 0 Pyridoxine. 0

group of lysine. Dialysis of the enzyme against 0.05 M Tris-HCl, pH 8.0, supplemented with 5 X 10-d M Cleland’s reagent and 5 x 10-d M EDTA, removes the pyridoxal phosphate and reduces the absorption at 410 to 420 rnp (Fig. 7). The activity of the purified enzyme is independent of added pyridoxal phosphate, but removal of the cofactor is accompanied by a significant in- activation of the enzyme. It is noteworthy that the pyridoxal phosphate remains associated with the enzyme if dialyzed against 0.05 M potassium phosphate, pH 7.5, supplemented with 5 X 1W4 M Cleland’s reagent and 5 X low4 M EDTA.

Table II presents the effects of various analogues of pyridoxal on the reactivation of the apoenzyme. Of the compounds tested, only pyridoxal phosphate and pyridoxamine phosphate served as cofactors. The reactivation of the dialyzed enzyme by these compounds occurs only after an appreciable lag. The lag with pyridoxamine phosphate is more pronounced than that with pyridoxal phosphate. This difference could possibly represent the time required for the enzyme to deaminate the pyridoxa- mine phosphate; this point is currently under investigation.

The affiity of the apoenzyme for pyridoxal phosphate was determined. Fig. 8 is a double reciprocal plot of the rate of the deaminase reaction as a function of pyridoxal phosphate con- centration. The K, of the enzyme for the cofactor as deter- mined from this plot is 1.2 X 10e6 M.

Cation Requirements

The purified enzyme showed no requirement for divalent cat- ions, and chelating agents such as CY,CY’-dipyridyl, EDTA, and o-phenanthroline at concentrations of 10m3 M had no effect on enzyme activity. Monovalent cations were only slightly stimu- latory; NH&l, KCl, NaCl, and LiCl, the only salts tested, each showed maximal stimulation (about 20%) at a concentration of 0.1 M in the assay.

Determination of Amount of Pyridoxal Phosphate Bound to Enzyme

The fact that pyridoxal phosphate remains bound to the en- zyme molecule during the course of the purification procedure facilitates determination of the valency of the enzyme with re- spect to the cofactor. Three independent methods were used to effect this determination. The first method was that of Yunis, Fischer, and Krebs (24). Threonine deaminase (6.9 mg) in 1.5 ml of 0.05 M potassium phosphate, pH 7.4, containing 8 X 10e4 M

L-isoleucine, 5 X 10m4 M EDTA, and 5 X 10V4 M Cleland’s re- agent, was added to 1.5 ml of 0.6 N perchloric acid; the mixture

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184 Threonine Deaminase from S. typhimurium. I Vol. 243, No. 1

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.6-

VPyridoxal Phosphate (f/&M) -

FIG. 8. The effect of pyridoxal phosphate concentration on the rate of the threonine deaminase reaction. Purified threonine deaminase was resolved of pyridoxal phosphate as described in the text. The calorimetric assay was used; a-ketobutyrate was deter- mined at 3-min intervals for 24 min, and the linear portion of the resulting curve was taken as the reaction velocity.

was allowed to stand at room temperature for 15 min and then centrifuged to remove the precipitated protein; finally, absorb- ance of the supernatant was measured at 295 rnp. By using the molar extinction coefficient of pyridoxal phosphate determined by Peterson and Sober (25), the quantity of pyridoxal phosphate was computed to be 0.66 pmole/6.9 mg of protein. This cor- responds to 0.97 mole of pyridoxal phosphate per 100,000 g of protein. Pyridoxal phosphate was also determined by the phenylhydrazine method of Wada and Snell (26). Purified threonine deaminase was treated with Ha04 in order to precipi- tate the protein (27). The supernatant was then assayed for pyridoxal phosphate. With this procedure it was found that 2.26 mg of enzyme contained 0.026 pmole of pyridoxal phosphate, or 100,000 g of enzyme contained 1.15 moles of pyridoxal phos- phate. The third method involved labeling the enzyme with radioactive pyridoxal phosphate. This was accomplished by purifying the enzyme from extracts obtained from cells grown on tritiated pyridoxine (see “Experimental Procedure”). Ideally, since the enzyme was prepared from an organism which was un- able to synthesize pyridoxal, the radiospecific activity of the pyridoxine added, corrected for the exchange of the label in the transformation of the pyridoxine to pyridoxal phosphate, should equal the radiospecific activity of the pyridoxal phosphate asso- ciated with the enzyme. However, this correction could prove to be invalid, owing to the uncertainty with regard to the degree of labeling of the various positions on the pyridoxine molecule, and so the radiospecific activity of the pyridoxal phosphate on the enzyme was independently measured. The tritiated pyri- doxal phosphate-labeled enzyme was prepared by the standard purification procedure. This preparation was pure by the cri- teria already mentioned. A portion of the enzyme from this preparation was diluted with a solution of 0.5% bovine serum albumin and placed in a boiling water bath for 10 min, the pre- cipitated protein was removed by centrifugation, and a portion of the supernatant was assayed for pyridoxal phosphate ac- cording to the tryptophanase procedure outlined in “Experimen- tal Procedure.” Control experiments with pyridoxal phosphate levels similar to those expected in the actual determination showed that 100% of the cofactor was recovered following the heating step. A portion of the supernatant obtained from the

heating step was counted for radioactivity, and the radiospecific activity was computed. The radioactivity and amount of pro- tein of another sample of the purified enzyme were measured. The previously computed radioactive specific activity and the previously determined extinction for the enzyme were used to determine the amount of pyridoxal phosphate and protein, re- spectively. The radiospecific activity of the holoenzyme was 94,000 cpm per mg of protein; the radiospecific activity of the pyridoxal phosphate was 11.1 x lo6 cpm per pmole; therefore, 1 mg of pure threonine deaminase contained 8.4 x 10m3 pmole of pyridoxal phosphate, or 100,000 g of enzyme contained 0.84 mole of pyridoxal phosphate.

The last procedure outlined above was designed in order to by- pass a major problem inherent in the other two procedures. The determination of the radiospecific activity of the pyridoxal phos- phate is independent of the efficacy of the heating procedure in releasing the cofactor from the enzyme and circumvents the ob- jection of incomplete resolution of the cofactor from the enzyme. Moreover, the enzymatic method possesses an advantage in that lower concentrations of pyridoxal phosphate can be determined and therefore smaller amounts of enzyme are consumed. Never- theless, the agreement between the independent determinations is good.

DISCUSSION

The method described for the purification of threonine deami- nase provides a relatively rapid and simple means for obtaining homogeneous preparations of this enzyme. Sufficient quantities of the enzyme for physical analyses were obtained by using con- ditions of cultivation which permit a maximal differential rate of synthesis of the enzyme by the bacterium; the specific activity of the crude extracts with respect to threonine deaminase was about 12 times that of crude extracts of the wild type strain. The en- zyme, which is relatively unstable when present in crude extracts, is stable when pure and concentrated. It was consistently ob- served in the course of the work described here that a milieu of high ionic strength provided unfavorable conditions with regard to the stability of the enzyme. This fact is probably related to the observation that high salt concentrations are effective in re- moving the homotropic interaction evident in Fig. 3. I f L-

isoleucine stabilizes the enzyme and promotes homotropic inter- actions by preventing exposure of the active center of the enzyme, then high salt concentrations that remove the homotropic inter- action may do so by exposing the active center, thereby also labilizing the enzyme.

Purified threonine deaminase was inhibited by isoleucine and exhibited the kinetic properties of cruder preparations of the en- zyme. Our results agree with those obtained by Maeba and Sanwal (15). These authors interpreted their results according to a kinetic model which was not based on structural considera- tions. However, the results can also be readily interpreted with reference to a definite quaternary structure. The reaction kinet- ics in the presence of low levels of isoleucine is of a higher order than in the absence of thii negative effector. By the model of Monod et al. (6), isoleucine can be visualized as bringing about homotropic interactions with respect to the substrate. This interaction can be eliminated specifically by L-valine or nonspecif- ically by high salt concentrations. Ammonium chloride, sodium chloride, potassium phosphate, sodium benzoate, and potassium acetate were al1 effective in removing the homotropic interaction. It is well known that salts alter the noncovalent interactions that

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Issue of January 10, 1968 R. 0. Burns and M. H. Zarlengo 185

are important in determining the tertiary and quaternary struc- ture of enzymes (28).

A subtle difference exists between the threonine deaminases of S. typhimurium and E. coli, in that the latter enzyme shows homotropic interactions in the absence of isoleucine (8). This difference suggests that whereas the enzyme studied here exists in a form in which the catalytic site is readily accessible, the E. coli enzyme exists in a form in which the active site is less avail- able.

In view of the allosteric nature of threonine deaminase, the effect of isoleucine in displacing the pH curve is not surprising, but, rather, fortifies the notion of the flexibility of the conforma- tion of this type of protein. It is interesting that in hemoglobin, in which it has been clearly demonstrated that conformational alterations accompany the binding of oxygen to the protein (29), a similar situation exists; increased acidity or pcoz causes a shift in the dissociation curve of oxyhemoglobin to the right. This “Bohr effect” in hemoglobin is at least partly explained by the effect of hydrogen ion concentration on the dissociation of an ionizable group (or groups) indirectly involved in the reaction of O2 with the hemoglobm molecule. The shift in the pH curve for threonine deaminase could be explained in comparable terms; that is, a certain group must be nonprotonated in order for en- zymatic activity to occur, and the binding of isoleucine to the en- zyme causes a shift in the pK of this group from 7.6 to 8.7 (if the inflections in the curves presented in Fig. 5 can be interpreted in this way). Such a shift in pK might be caused by a reorienta- tion of the pertinent group within the enzyme molecule. An alternative explanation of this observation is that the binding of isoleucine to the enzyme is directly dependent on the ionic state of the binding site per se; i.e. the binding site for isoleucine would have to be protonated. The two alternatives ought to be dis- tinguishable, since in the first case the effect would be reciprocal and the binding of isoleucine would be accompanied by an uptake of protons.

Pyridoxal phosphate was shown to be associated with the purified enzyme. The spectrum shown in Fig. 7 suggests that this cofactor is associated with the enzyme by way of an aldamine bond (30). Precedent would suggest that the e-amino group of lysine is involved, but no direct proof of this involvement is available. Pyridoxal phosphate or pyridoxamine phosphate was capable of restoring activity to the enzyme following treatment under conditions intended to remove the cofactor. Although these observations suggest that pyridoxal phosphate is in fact a catalytic cofactor for the p elimination reaction involved in the transformation of L-threonine to cr-ketobutyrate, they do not rule out the possibility that pyridoxal phosphate is also involved in maintaining the structural integrity of the enzyme. This point is currently being investigated. It should also be mentioned that treatment of the purified enzyme with borohydride or isoniazid destroys catalytic activity.’

Three independent methods were used to determine the amount of pyridoxal phosphate bound to the enzyme. The average value of the three determinations is 0.99 mole of pyri- doxal phosphate per 100,000 g of enzyme. Therefore, the mini- mum molecular weight of the enzyme is 101,000. However, the sedimentation coefficient of the pure enzyme, 8.7 x lo-i3 cm

1 R. 0. Burns, unpublished observations.

per set, indicates that the molecular weight is much higher. It is more probable that the enzyme contains 2 eq of pyridoxal phos- phate and weighs closer to 200,000. This is consistent with re- sults (mol wt 196,000) presented in the following paper (21).

I f pyridoxal phosphate is in fact a component of the active,site of the enzyme, an assumption which at this point seems highly likely, then threonine deaminase would be a structure con- taining two active sites. Such a structure lends credence to the model discussed here.

Acknowledgments-The authors wish to thank Mrs. Karen K. Goodrich for her expert assistance in this work. One of us (R. 0. B.) wishes to thank Professor H. E. Umbarger of Purdue University for introduction to this problem, as well as for his con- tinued interest and stimulation in these endeavors.

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R. O. Burns and Mario H. ZarlengoPROPERTIES

: I. PURIFICATION ANDSalmonella typhimuriumThreonine Deaminase from

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