Threonine Deaminase from SaZmoneZZa typhimurium EFFECT OF REGULATORY LIGANDS ON THE BINDING OF SUBSTRATES AND SUBSTRATE ANALOGUES TO THE ACTIVE SITES AND THE DIFFERENTIATION OF THE ACTIVATOR AND INHIBITOR SITES FROM THE ACTIVE SITES*

deaminase, EC displays a positive circular dichroism with a maximum rotation at 410 nm owing to the aldimine-bonded cofactor pyridoxal 5’-monophosphate. The substrates, Ahreonine, t-allothreo-nine, and L-serine, and the competitive inhibitors of enzyme activity, o-threonine, o-allothreonine, and o-serine, cause a loss of circular dichroism presumably because of transfer of the aldimine bond from the protein to these ligands. The “allosteric” effector ligands, L-isoleucine and L-valine, have no effect on the optical activity of the enzyme. By employ- ing these features of the enzyme, it is shown that Gsoleu-tine prevents the Ahreonine-and L-allothreonine-imparted loss of circular dichroism but does not prevent the D-threo-nine-, o-allothreonine-, o-serine-, or L-serine-imparted loss of optical activity. These results imply that the inhibition of enzyme activity by IAsoleucine, depending

The substrates, Ahreonine, t-allothreonine, and L-serine, and the competitive inhibitors of enzyme activity, o-threonine, o-allothreonine, and o-serine, cause a loss of circular dichroism presumably because of transfer of the aldimine bond from the protein to these ligands. The "allosteric" effector ligands, L-isoleucine and L-valine, have no effect on the optical activity of the enzyme. By employing these features of the enzyme, it is shown that Gsoleutine prevents the Ahreonine-and L-allothreonine-imparted loss of circular dichroism but does not prevent the D-threonine-, o-allothreonine-, o-serine-, or L-serine-imparted loss of optical activity. These results imply that the inhibition of enzyme activity by IAsoleucine, depending upon the substrate, is explicable in terms of perturbation of either initial substrate binding or the catalytic mechanism per se, i.e. Asoleucine exerts a K effect when either t-threonine or t,-allothreonine serves as substrate but a V effect when Lserine is substrate. These results also definitively demonstrate the separation of the inhibitor and active sites of biosynthetic Ahreonine deaminase. The separation of the activator site and active sites of biosynthetic threonine deaminase is demonstrated by the inability of L-valine to compete with o-threonine in causing loss of circular dichroism. It is shown that L-serine mimics Ahreonine as a substrate for Ahreonine deaminase, i.e. the reaction velocity becomes a high order function of Lserine concentration in the presence of Asoleucine. Loss of circular dichroism in the presence of L-isoleucine, however, is a first order function of L-serine concentration. These results together with the observation that L-valine overcomes isoleucine inhibition of the enzyme reaction with L-serine as substrate show that the modulation of the activity of Ahreonine deaminase is wrought by the interaction * This work was supported by Grant GM-12551 from the National Institutes of General Medical Sciences. This paper is the fifth in the series on threonine deaminase from Salmonella typhimurium. The preceding paper in this series is Ref. 3. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ United States Public Health Service Trainee. of activator and inhibitor sites and also demonstrates that the activator site is separate from the active sites. portions of toxin and catalase were added and the reaction allowed to proceed overnight.
In both cases, the formation of u-ketobutyrate was monitored as the 2,4-dinitrophenyl hydrazone (81. Each mixture was treated as follows. The mixtures were adjusted to pH 6.5 with dilute HCl and loaded onto a column of Dowex AG50W-8x (50 to 100 mesh). The column was then washed with water until the effluent contained no cu-keto acid and the amino acid was eluted with 1 N ammonium hydroxide.
The eluent was evaporated to dryness and the residue dissolved in a minimum volume of hot water; the amino acid was crystallized by addition of ethanol (7 volumes) and the crystals were dried with ether.
The samples were recycled through the entire resolution procedure and finally checked for optical purity by the use of the n-amino acid oxidase and the venom. Thin layer chromatography of the resolved samples on microcrystalline cellulose plates, developed with a solvent system containing lbutanol:methylethylketone:H,O:NH,OH (5:2:1:11 and which is known to resolve allothreonine and threonine (91, shows that each sample is approximately 5% contaminated with threonine.
Kinetic Parameters for L-Allothreonine-The K,,! for L-allothreonine was estimated by measuring the tangential slope of a continuous reaction at the concentrations of L-allothreonine shown in Fig.  6. This procedure was made necessary by the apparent low affinity of the enzyme for L-allothreonine as well as by an appreciable level (-5%) of contamination with L-threonine. The reaction mixture contained 0.1 rnM potassium phosphate, pH 7.5, and the reaction was initiated with 8 rnsr L-allothreonine. The reaction was allowed to proceed until the contaminating L-threonine was depleted as shown by thin layer chromatography (91 of a duplicate reaction mixture.
The concentration of L-allothreonine at various times during the reaction was calculated by difference; the reaction was allowed to go to completion and the total concentration of L-allothreonine present following depletion of the L-threonine was determined by using the relationship that one absorbance unit at 235 nm equals 5.6 pmol of a-ketobutyrate.

Effect
of L-Zsoleucine and L-Valine on Circular Dichroic Spectrum of L-Threonine As in the case of L-threonine, the transfer of the aldimine bond from the enzyme to n-threonine is reversible. A 200-fold dilution of the enzyme-n-threonine mixture into reaction mixture containing L-threonine as substrate with and without added pyridoxal 5'-monophosphate resulted in identical rates of catalysis; this also shows that the loss of optical activity is not caused by a n-threonine-promoted resolution of cofactor from the enzyme. indicates that n-threonine is a dead-end competitive inhibitor of biosynthetic L-threonine deaminase.' The data depicted in Fig.  4A show that inhibition of activity is a first order function of n-threonine concentration; this observation is consistent with n-threonine acting at the active site rather than the inhibitor (L-isoleucine) site because inhibition of enzyme activity by L-isoleucine is a second order function of concentration (3). The K, computed from these data is 7.3 (51.9) mM. Fig. 5  i.e. the affinity of o-allothreonine for the enzyme is 3 orders of magnitude greater than is that of Lallothreonine.
The ability of the D and L isomers of allothreonine to cause the loss of 300 to 500 nm circular dichroism of L-threonine deaminase was tested. These compounds exhibit the same properties as do the threonine isomers; L-allothreonine causes an immediate loss of optical activity which returns as the substrate is removed by the action of the enzyme and Dallothreonine causes a loss which is typical of that seen with o-threonine.
The ability of L-isoleucine to prevent the allothreonine-imparted loss of optical activity was tested; Lisoleucine prevents the loss of optical activity caused by Lallothreonine, whereas it does not prevent the loss of optical activity caused by n-allothreonine. Presumably, L-isoleucine prevents the binding of L-allothreonine, but not the binding of n-allothreonine to the active site. Fig. 7 shows the loss of circular dichroism as a function of n-allothreonine concentration; the K, determined from these data is 0.61 (kO.31) mM. These results are identical in the presence or absence of 2 mM L-isoleucine, as is shown in Fig. 7  The maximal rate of conversion of L-serine to pyruvate is the same as that for conversion of L-threonine to w ketobutyrate.
The major difference is that L-serine does not bind as well as L-threonine to either the active sites or the activator site; the K,,, and the K,, are approximately 20-fold higher, 90 and 55 mM, respectively (3). As might be expected from t,he inhibitor properties of the D isomers of threonine and allothreonine, n-serine also was shown to be a competitive inhibitor of enzyme act.ivity.' Fig. 4C shows that inhibition is a first order function of n-serine concentration with a K, of 2.4 (k1.0) mM. As in the case with the other D isomers, n-serine causes a loss of optical activity and this loss is not prevented by L-isoleucine. Fig. 9 shows the effect of n-serine on the circular dichroism of L-threonine deaminase at, 410 nm. The K,, calculated from these data is 3.7 (kO.7) mM. Titration of the enzyme with L-isoleucine, at concentrations up to 2 mM, was unable to cause a regain in the optical activity in the presence of n-serine. As in the cases of the other two substrates, L-serine causes a complete loss of circular dichroism which is restored as the substrate is enzymatically removed. Unlike with the other substrates, however, L-isoleucine does not prevent loss of optical activity. Fig. 10 shows the effect of L-serine on the optical activity of L-threonine deaminase in the presence of 4 mM L-isoleucine; the K,, for L-serine, calculated from these data, is 147 (*14) mM.

Differentiation of Activator and Inhibitor Sites from Active Sites of Biosynthetic
L-Threonine Deaminase -The results presented in Fig. 2 show that neither L-valine nor L-isoleucine causes the loss of the circular dichroism of threonine deaminase. Also, L-isoleucine does not prevent the loss of circular  Fig. 11, although failing to reveal the Threonine Deaminase Regulatory Sites-Active Sites Interaction basis for the observed behavior of the various compounds, nonetheless, do reveal features of possible significance. The models of L-threonine, L-allot hreonine, and L-serine show identical orientation of the amino, carboxyl, and alcohol functions which may be related to the substrate nature of these compounds. Comparison of the K,,, values for these substrates, as listed in Table I, suggests that the orientation of the methyl group of L-threonine facilitates the binding of this compound. The substrates, L-serine and L-allothreonine, have K,,, values considerably higher than the one for L-threonine. Fig. 11 shows L-serine and L-allothreonine oriented in a way to appear remarkably similar with the methyl group of L-allot hreonine partially eclipsed so as to appear like L-serine which lacks a methyl group. This line of reasoning appears to be inconsistent with the K,, and K, values for n-serine and Dallothreonine which are nearly 2 orders of magnitude less than those for the respective L isomers and where the sole similarity appears to be the orientation of the amino and hydroxyl groups. The basis for the relatively avid binding of the n isomers is not clear. It is unlikely that the K,, values for L-serine and L-threonine do not reflect the true K,, values for these compounds. The direct measurement of the K,, for Lserine in the presence of L-isoleucine (Fig. 10) as measured by loss of circular dichroism gives a value similar to the K,,,. Also, the observation that t hreonine deaminase is inactive on the racemic mixture of allot hreonine is consistent with the K,, of L-allot hreonine being considerably higher than the K,, for D-allothreonine which was measured directly. A possible basis for the avid binding of the D isomers is the orientation of the carboxylat e ion which may be capable of electrostatic int eraction with the protonated amino group which is made available on the protein as a consequence oft he transfer oft he aldimine bond from the protein to the amino group of the inhibitor.
The differential K and V (4) effect must be based in the structural attributes of the threonines and serines as well as in the conformational change in the protein exerted by Lisoleucine. The conformat ional change which prevents the catalytic function of the active site could simultaneously result in steric hindrance to those compounds with /3 substituents which are properly oriented relative to the functional binding groups on the molecules. Space filling models of the compounds under consideration show that the methyl group of L-threonine and L-allothreonine could provide hindrance to the intrusion of these molecules into the active sites when the enzyme is in the catalytically inactive conformation. Serine lacks the methyl group and n-threonine and n-allothreonine can be pictured as having methyl groups oriented away from the binding plane of these molecules. A separate line of evidence is available to suggest a role for p substituents in binding to biosynthetic L-threonine deaminase.' Two altered biosynthetic threonine deaminases have been purified from mutants of Salmonella typhimurium which were selected on the basis of containing feedback negative threonine deaminases. These enzymes, in addition to showing a decreased sensitivity to inhibition by L-isoleucine, also show a lo-fold increase in K,,, for L-threonine but a slight decrease in K,,, for L-serine. These results point either to a perturbation of a methyl group binding function or to a weaker version of the steric exclusion of the methyl group which, as pointed out above, may be linked functions.
The data presented in Table I present a subtle but interesting contrast between those values which were determined as the basis of enzymatic activity (K, and K,,,) and those based upon loss of circular dichroism (K,). It is of interest to note that the K, values which were measured by change in a physical property are approximately 2-fold higher than the K, values (as well as the one K,n where the comparison is available). The K, values, K,,,, and K,, values were determined under the same conditions of ionic strength and temperature with the sole difference in conditions being the enzyme concentration employed; the concentration of enzyme in the K,, measurements was 3 to 4 orders of magnitude greater than that used in typical enzyme activity measurements. Although the large difference in enzyme concentration may be the basis for the observed differences, other factors also must be considered. It is well established that Michaelis' constants do not necessarily correspond to dissociation constants and this or the fact that the K,, for L-serine was determined in the presence of L-isoleucine could explain the difference between the K,,, and Kd for L-serine. The situation with the competitive inhibitors, however, is different since K, and K,, values should be equal. A possible explanation for the observed difference in the two sets of parameters is a type of "half of the site" activity (14). In the case of the competitive inhibitors, the liganding of one of the two active sites could be sufficient to inhibit the enzyme and in the case of the substrate, although both active sit.es are capable of binding ligand, only one of the sites could be catalytically active. In contrast, if the loss of circular dichroism is the result of aldimine bond transfer, then both pyridoxal 5'-monophosphat es must be transferred in order to obtain total loss of opt ical activity. According to these speculations, the enzyme activity employed t.o det.ermine the K,,, and K, would be the expression of only one of two equivalent sites each with the same probability of liganding the effect,or molecule, whereas in the case of the K,, measurements, t,he loss of circular dichroism is a function of the liganding of both sites. Therefore, probability considerations dictate that since t.here are two ways to realize a single event when measuring K, or K,,, and only one way to decrease the circular dichroism, then the K, and K,,, values should be approximately one-half of t,he K,, values. Efforts are currently under way to determine whether or not a type of half sit,e activity is, in fact, operative in t.hreonine deaminase.
The results presented in the present invest.igat,ion provide additional insight into the relat.ionship among the stereospecific sites on biosynthetic r.-threonine deaminase. The inability of L-valine to compete with n-threonine in binding to the active site (Fig. 5) shows that the activator sit.e and active sit.es are separat.e. This is further supported by the observation t.hat n-t.hreonine does not. compete with L-valine in direct. binding experiment,s.' The results of the kinet.ic analysis and the circular dichroism measurements with L-serine provide proof of the allosteric nature of L-threonine deaminase as well as the existence of an activator site. The inability of Lisoleucine to prevent the binding of L-serine but, yet, prevent its enzymatic conversion to pyruvate and ammonia shows that the inhibitor sites are separate from the active sites. The observation that, the loss of circular dichroism is a first. order function of L-serine concentration in the presence of L-isoleutine, whereas the rate of the enzyme reaction in the presence z R. 0. Burns, unpublished observations. of L-isoleucine is a high order function of L-serine concentration shows that L-serine must be binding to a site (i.e. the activator site) other than the active site in order to overcome L-isoleucine inhibit ion. Achnowledgrnents -We wish to thank Dorothy Thompson, Douglas Erickson, and Richard Rosenberg for t ethnical assistante and Donna Crutchfield for the typing of this manuscript. We would also like to thank Drs. Charles Tanford and Jacqueline Reynolds for use of some of their laboratory facilities and Dr. Y. Nozaki for instructions in circular dichroism instrumentation.