Cloning, expression, purification, and characterization of biosynthetic threonine deaminase from Escherichia coli.

Feedback inhibition of the regulatory enzyme threonine deaminase by isoleucine provides an important level of enzymic control over branched chain amino acid biosynthesis in Escherichia coli. Cloning ilvA, the structural gene for threonine deaminase, under control of the trc promoter results in expression of active enzyme upon induction by isopropyl 1-thio-beta-D-galactoside to levels of approximately 20% of the soluble protein in cell extracts. High level expression of threonine deaminase has facilitated the development of a rapid and efficient protocol for the purification of gram quantities of enzyme with a specific activity 3-fold greater than previous preparations. The catalytic activity of threonine deaminase is absolutely dependent on the presence of pyridoxal phosphate, and the tetrameric molecule is isolated containing 1 mol of cofactor/56,000-Da chain. Wild-type threonine deaminase demonstrates a sigmoidal dependence of initial velocity on threonine concentration in the absence of isoleucine, consistent with a substrate-promoted conversion of the enzyme from a low activity to a high activity conformation. The enzymic dehydration of threonine to alpha-ketobutyrate measured by steady-state kinetics, performed at 20 degrees C in 0.05 M potassium phosphate, pH 7.5, is described by a Hill coefficient, nH, of 2.3 and a K0.5 of 8.0 mM. The negative allosteric effector L-isoleucine strongly inhibits the enzyme, yielding a value for nH of 3.9 and K0.5 of 74 mM whereas enzyme activity is greatly increased by L-valine, which yields nearly hyperbolic kinetics characterized by a value for nH of 1.0 and a K0.5 of 5.7 mM. Thus, these effectors promote dramatic and opposing effects on the transition from the low activity to the high activity conformation of the tetrameric enzyme.

Feedback inhibition'of the regulatory enzyme threonine deaminase by isoleucine provides an important level of enzymic control over branched chain amino acid biosynthesis in Escherichia coli. Cloning iluA, the structural gene for threonine deaminase, under control of the trc promoter results in expression of active enzyme upon induction by isopropyl I-thio-fi-D-galactoside to levels of approximately 20% of the soluble protein in cell extracts. High level expression of threonine deaminase has facilitated the development of a rapid and efficient protocol for the purification of gram quantities of enzyme with a specific activity %fold greater than previous preparations. The catalytic activity of threonine deaminase is absolutely dependent on the presence of pyridoxal phosphate, and the tetrameric molecule is isolated containing 1 mol of cofactor/ 56,000-Da chain. Wild-type threonine deaminase demonstrates a sigmoidal dependence of initial velocity on threonine concentration in the absence of isoleucine, consistent with a substrate-promoted conversion of the enzyme from a low activity to a high activity conformation. The enzymic dehydration of threonine to aketobutyrate measured by steady-state kinetics, performed at 20 "C in 0.05 M potassium phosphate, pH 7.5, is described by a Hill coefficient, nH, of 2.3 and a of 8.0 mM. The negative allosteric effector L-isoleucine strongly inhibits the enzyme, yielding a value for nH of 3.9 and of 74 mM whereas enzyme activity is greatly increased by L-valine, which yields nearly hyperbolic kinetics characterized by a value for nH of 1.0 and a of 5.7 mM. Thus, these effectors promote dramatic and opposing effects on the transition from the low activity to the high activity conformation of the tetrameric enzyme.
Control of metabolic pathways by end product inhibition of allosteric enzymes is a fundamental paradigm of biochemistry (Stryer, 1988). Enzymic control of branched chain amino acid biosynthesis in Escherichia coli is achieved in part by the regulation of allosteric threonine deaminase (threonine dehydratase; L-threonine hydro-lyase (deaminating); EC 4.2.1.16) (Umbarger, 1987). Threonine deaminase, composed of four identical polypeptide chains of 56,202 daltons (Lawther et al., 1987;Cox et al., 19871, catalyzes the first committed step in the biosynthesis of isoleucine, the pyridoxal phosphate-dependent dehydration/deamination of threonine to a-ketobutyrate and ammonia. Early enzymological studies on biosynthetic threonine deaminase using crude cell lysates (Umbarger, 1956;Brown, 1956, 1957a, 195713;Changeux, 1961Changeux, , 1962Changeux, ,1963Freundlich and Umbarger, 1963) provided initial experimental support for the notion that different conformations of regulatory enzymes could be stabilized by ligand binding to sites distinct from their active sites, and these studies were influential in the development of the seminal concerted model for allosteric regulation (Monod et al., 1965). This initial work showed that threonine deaminase was inhibited by isoleucine, the end product of the pathway, whereas a stimulation of activity was observed in the presence of valine, the prouct of an essentially parallel biosynthetic pathway. Although later work with purified enzyme from E. coli K-12 demonstrated a hyerbolic dependence of activity on threonine concentration, catalysis was inhibited by isoleucine, and this inhibition could be reversed by valine (Calhoun et al., 1973;Koerner et al., 1975).
The molecular basis for control by feedback inhibition poses a central question in analyses of the cooperative behavior of proteins: how ligand binding at one site of an oligomer promotes changes in ligand affinity at another site. In an effort to study the protein interactions that regulate enzyme activity in threonine deaminase, the structural gene for threonine deaminase, iluA, has been cloned into plasmid vectors for mutagenesis and expression of the enzyme. High level expression of threonine deaminase from the trc promoter has facilitated the development of a rapid and efficient protocol for the purification of large quantities of enzyme of high specific activity. Interestingly, steady-state kinetic studies of the highly purified enzyme have revealed a sigmoidal dependence of the initial velocity on threonihe concentration in the absence of isoleucine, in contrast to some previous reports. The heterotropic effectors isoleucine and valine markedly shift the cooperative kinetics of the enzyme, promoting almost complete inhibition and activation, respectively. This work provides the groundwork for elucidation of the kinetic, thermodynamic, and structural features of threonine deaminase responsible for cooperativity and feedback inhibition.

EXPERIMENTAL PROCEDURES AND RESULTS'
Steady-state Kinetics-Since several biochemical attributes of threonine deaminase from E. coli K-12 had been character-' Portions of this paper (including "Experimental Procedures," part of "Results," Figs. 1-4, and Table 11) are presented in miniprint at the end of this paper. The abbreviations used are: SDS, sodium dodecyl sulfate; kb, kilobase. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press. 5801 ized previously using enzyme purified in small quantities (Calhoun et al., 1973;Koerner et al., 1975), one of the first goals after expressing the enzyme and purifying it using a newly developed protocol was to reproduce the kinetic properties of the enzyme, especially with respect to the effects of isoleucine and valine on saturation curves. This was of additional interest since the specific activity of the enzyme isolated from the new procedure was approximately 3 times that reported previously. As can be seen in Fig. 5A, the dependence of initial velocity on threonine concentration for threonine deaminase is markedly sigmoidal, in sharp contrast to that observed previously. This surprising result is shown even more clearly in Fig. 5B, a sensitive Eadie plot, which is concave downward, indicating positive homotropic cooperativity (Hensley et al., 1981). Analysis of these data as described under "Experimental Procedures" yields a value for the maximum velocity, V,,,, of 214 pmol/mg/min, a Hill coefficient, nH, of 2.3, and a of 8.0 mM. The effect of the heterotropic effectors isoleucine and valine similarly were determined in 50 mM potassium phosphate, pH 7.5, and at 20 "C. The effect of isoleucine on the steady-state kinetics of threonine deaminase is shown in Fig. 6. As can be seen in Fig. 6A, isoleucine markedly inhibits the activity of the enzyme, especially a t threonine concentrations around the (8.0 mM) observed in the absence of effectors. This inhibition is reflected in the Eadie plot of Fig. 6B by a shift in the maximum of the curve to higher saturation (Dahlquist, 1978). This almost total inhibition of enzyme activity at low substrate concentration is also reflected in the kinetic parameters determined under these conditions. Data analysis yielded values for nH of 3.9, for Koa of 74 mM, and for V,,, of 180 pmol/mg/min. Conversely, the effect of valine was to activate the enzyme even in the absence of isoleucine. As can be seen in Fig. 7A, the effect of valine on the steady-state kinetics is to increase activity near the Ko.5 observed in the absence of effectors. This increase in activity gives rise to nearly hyperbolic kinetics, as can be seen in the Eadie plot in  data yields values for nH of 1.0, for K 0 . 5 of 5.7 mM, and for V,,, of 225 pmol/mg/min.

DISCUSSION
The regulation of catalytic activity in allosteric enzymes is mediated by conformational changes upon ligand binding which alter the free energy of interaction between subunits. In an effort to study these interactions in biosynthetic threonine deaminase from E. coli, the structural gene encoding this enzyme, iluA, was modified and cloned into plasmid vectors for convenient mutagenesis of the gene and expression of the enzyme. The strategy used to achieve this aim is summarized in Fig. 1. Modification of iluA by site-directed mutagenesis to engineer restriction enzyme sites at the initiation and termination codons in the gene resulted in a "cas-sette" containing only iluA-coding sequences which was used to produce authentic enzyme. Cloning the ilvA cassette into pUC120 facilitated production of single-strand DNA for sequencing experiments, yielding excellent agreement with previously published reports of the nucleotide sequence for this region of the E. coli ilv operon (Lawther et al., 1987;Cox et al., 1987). Induction of enzyme synthesis by the addition of isopropyl 1-thio-6-D-galactoside to midlog cells containing the iluA cassette cloned under control of the trc promoter in pKK233-2 resulted in expression of threonine deaminase to levels of about 20% of the soluble protein in crude cell extracts. This was demonstrated by subjecting cell extracts to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. As can be seen in Fig. 2, a band of M , 56,000 can be detected in the lane corresponding to induced cells containing pEE6, consistent with the molecular weight of chains of threonine deaminase of 56,202.
Threonine deaminase has been purified from a number of sources, including E. coli, but only in small quantities (Calhoun et al., 1973;Koerner et al., 1975). High level expression of threonine deaminase has facilitated the development of a protocol for the rapid purification of large quantities of highly active enzyme. Efficient purification is achieved in four stages, involving three column chromatography steps, and yields homogeneous enzyme in a 50% overall yield. A summary of this protocol and the yield of enzyme at each step is presented in Table I. The homogeneity of the enzyme purified using this procedure is demonstrated in the sodium dodecyl sulfatepolyacrylamide gel shown in Fig. 3, which exhibits a single band of 56,000 Da. N-terminal amino acid analysis of the first 1 2 residues of threonine deaminase was in agreement with that predicted from the nucleotide sequence of the iluA gene.
The specific activity of threonine deaminase purified using this new protocol is 210 gmol/mg/min at 50 mM threonine in 50 mM potassium phosphate, pH 7.5, at 20 "C. When the enzyme was assayed at 37 "C, at elevated pH, or in the presence of ammonium ion, an increase in enzyme activity was observed. A direct comparison of enzyme isolated in this study with that in other investigations was achieved by conducting assays in 0.1 M potassium phosphate, pH 8.0, 0.1 M ammonium chloride, at 37 "C. The specific activity determined under these conditions was 630 units/mg/min, corresponding to a 3-fold increase over that reported previously (Calhoun et al., 1973;Koerner et al., 1975). Hence, the rapid and efficient purification protocol made possible by the high level expression of threonine deaminase has resulted in homogeneous enzyme of high specific activity. A summary of the effect of temperature and buffer conditions on the steady-state kinetics of threonine deaminase is presented in Table 11.
Since the specific activity of threonine deaminase prepared as described above was 3 times higher than previous preparations, it was of interest to investigate the steady-state kinetics of the enzyme-catalyzed reaction to see how they compared with previous results. As can be seen in Fig. 5A, the initial velocity for threonine deaminase demonstrates a sigmoidal dependence on threonine concentration. This is shown dramatically in Fig. 5B, a sensitive Eadie plot, which is concave downward indicative of positive homotropic cooperativity (Hensley et al., 1981). Analysis of the cooperative kinetics data as described under "Experimental Procedures" yields a value for V, , , of 214 units/mg/min, a value for n~ of 2.3, and a KO.:, of 8.0 mM. Although several early studies using either crude cell lysates or partially purified enzyme showed (to varying degree) sigmoidal kinetics in enzyme assays, later work using purified threonine deaminase from E. coli K-12 showed a hyperbolic dependence of initial velocity on threonine concentration, in sharp contrast to results obtained in this study (Umbarger, 1973(Umbarger, , 1987. Cooperativity in steadystate kinetics has been observed on several different preparations of enzyme, using two different assays, and under several different experimental conditions. Indeed, under experimental conditions identical with respect to temperature and buffer composition to those used in previous investigations, cooperative kinetics were still observed. This is illustrated in Fig. 4A, which shows a saturation curve for steadystate kinetics in 0.1 M potassium phosphate, pH 8.0, 0.1 M ammonium chloride, at 37 "C. Analysis of this data yielded kinetic parameters for nH of 1.6, and for KO.:, of 3.8 mM, and positive cooperativity is seen readily in the Eadie plot of Fig.

4B.
Given the dramatic differences in kinetic properties in the absence of ligands that affect the activity of threonine deaminase, it was of interest to evaluate the effects of isoleucine and valine on the activity of the enzyme. The effect of isoleucine on the sigmoidal kinetics of threonine deaminase can be seen in Fig. 6. Isoleucine strongly inhibits the enzyme, virtually eliminating activity at threonine concentrations near the observed in the absence of effectors. This negative allosteric effector also increases the cooperativity of the enzyme as seen in Fig. 6B, an Eadie plot, for which the maximum of the curve is shifted to higher saturation (Dahlquist, 1978). The kinetic parameters for threonine deaminase assayed in 50 mM potassium phosphate, pH 7.5, at 20 "C in the presence  (Bradford, 1976).
Specific activity was determined a t 50 mM threonine in 50 mM potassium phosphate, pH 7.5, a t 20 "C using Protein concentration was determined using an extinction coefficient for threonine deaminase of 0.74 cm2 mg" the assays described under "Experimental Procedures." a t 280 nm. of 50 p M isoleucine are a nH of 3.9 and a K0.5 of 74 mM. The effect of valine is to activate the enzyme at threonine concentrations near the as can be seen in Fig. 7. Addition of valine to assays conducted in phosphate buffer at 20 "C results in nearly hyperbolic curves, as can be seen in the Eadie plot of Fig. 7B. Kinetic parameters under these conditions yield a value for n~ of 1.0 and a Ko.5 of 5.7 mM. Although the values for maximal velocities in the absence and presence of valine, 214 versus 225 pmol/mg/min, respectively, are nearly identical, the value obtained in the presence of isoleucine, 180 pmol/mg/min, was slightly lower. It thus appears that valine acts to reduce cooperativity and whereas isoleucine increases cooperativity and KO& and neither has a significant effect on the maximal velocity.
A second interesting feature of the steady-state kinetics concerns the values determined for the Hill coefficients. It is well known that the value for a Hill coefficient for cooperative ligand binding to a protein cannot exceed the number of binding sites (Dahlquist, 1978). Steady-state kinetics of threonine deaminase performed in phosphate buffer, pH 7.5, at 20 "C yield Hill coefficients of 2.3 in the absence of effectors, 1.0 in the presence of valine, and 3.9 in the presence of isoleucine, suggesting at least four functional active sites. However, a previous investigation of the pyridoxal phosphate content in threonine deaminase concluded that only two sites for the cofactor were present in the tetrameric molecule (Koerner et al., 1975). Thus, other preparations of threonine deaminase apparently contained only two functional active sites, in contrast to that expected from the kinetic analysis of highly expressed enzyme purified with a new protocol. This prompted a reinvestigation of the pyridoxal phosphate stoichiometry of threonine deaminase. Two different methods were used on a number of preparations of enzyme, both revealing the presence of 1 mol of pyridoxal phosphate/ 56,000-Da chain, or four functional active sites/tetrameric enzyme molecule. This result is supported fully by analysis of the kinetic properties of the enzyme and may suggest a possible source for the discrepancy in specific activities observed in this study relative to other work. Moreover, it is worthwhile to note that the effects of isoleucine and valine on the activity of threonine deaminase in phosphate buffer, pH 7.5, at 20 "C are exceptionally strong. In the presence of valine, virtually hyperbolic kinetics are observed, indicating nearly maximal activation by this ligand under these conditions. Conversely, isoleucine gives rise to pronounced inhibition, characterized by a Hill coefficient of 3.9 (of a maximum and valine promote dramatic and opposite changes in the enzyme kinetics of threonine deaminase which may, therefore, provide an advantage in a detailed investigation of the molecular basis of their action. Previous kinetics studies on biosynthetic threonine deaminase from E. coli (Hatfield, 1971;Calhoun et al., 1973) and Salmonella typhimurium (Hatfield and Umbarger, 1968;Burns and Zarlengo, 1968;Hatfield and Burns, 1970;Burns, 1971) were performed in buffers containing 0.1 M ammonium chloride since its presence increased enzyme activity. The buffer conditions used in this study excluded ammonium chloride, despite its effect of increasing enzyme activity, for several reasons. First, ammonium ion is one of the final products of the reaction and therefore may have some, although presumably low, affinity for the active site. Also, ammonium ion may possibly form a Schiff base with pyridoxal phosphate, as has been reported for tryptophanase, for example, in the first step of enzyme-catalyzed tryptophan synthesis from indole, pyruvate, and ammonia (Watanabe and Snell, 1972). Finally, many enzymes that require pyridoxal phosphate for catalysis are activated either by ammonia or monovalent cations (Hatanaka et al., 1962;Suelter, 1970;Snell, 1975;Toraya et al., 1976). Therefore, no additional exogenous ions (other than buffer counterions) were included in buffers for this study. This attempt was made since in ongoing studies of enzyme kinetics and other biochemical properties of threonine deaminase the need to consider the interaction of inhibiting or activating ions with wild-type and mutant enzymes would be minimized. However, in light of previous work and to determine if the presence of monovalent cations could account for some of the differences seen in this study relative to others, it was of interest to determine the effect of ammonium chloride on the activity of threonine deaminase. As can be seen in Fig. 8, the addition of ammonium ion led to a marked alteration of the activity of the enzyme at low (2 mM) threonine concentration. A maximum increase in activity of 50% was observed between 0.08 and 0.12 M ammonium chloride. However, increasing ion concentration beyond the activation range gives rise to inhibition of activity below that seen even in the absence of ammonium chloride. This behavior is reminiscent of the effect of the inhibitor succinate on the activity of aspartate transcarbamoylase (Gerhart and Pardee, 1963). The phemomenon has been attributed to substoichiometric binding of inhibitor to active sites at low inhibitor concentration, which promotes the transition from the low activity to high activity conformation, thereby increasing the activity of unligated active sites (Foote and Schachman, 1985). At higher inhibitor concentrations, almost complete inhibition is seen. It is tempting to speculate that ammonium ion plays a similar role in the activation and inhibition of threonine deaminase. The high concentration of ammonium ion necessary for activation of threonine deaminase probably reflects its poor affinity for the active site, and further experiments are necessary to test this hypothesis. This effect may also account in part for the hyperbolic kinetics observed for threonine deaminase in previous investigations. If enzyme activity was low because of decreased levels of cofactor, and ammonium ion binding to some active sites converted other sites to the high activity conformation, then Michaelian kinetics might have been observed. tryptone and yeast extract were from Difco. L-threonine, L-isoleucine and L-valine used in steady-state kinetics were from Fluka. All other chemicals were of reagent grade purity.
The first assay relied on coupling the formation of a.ketohutyrate to the oxidation of NADH by Threonine deaminase activity was measured by two different assays. enzyme during stages of purification. as well as for kinetics studies with homogeneous enzyme. lactate dehydrogenase essentially as described hy Bums (1971) and was used to assess yields of The second assay. used only with purified enzyme. measured directly the formation of a-ketohutyrate at 230 nm on a Shimadtu UV-265 recording spec~opho[ometer (Davis. 1965). An exp.rimentally determined value for the extinction coefficient of a-ketobutyrate in 50 mM potassium phosphate, pH 7.S at 230 nm was 540 M-1 cm-1, in excellent agreement with a conducted as described in figure legends. prcvious determination by Feldberg and Datta (1971). Assays for kinetic studies were m D e s r m i n a t i o n : Protein concentration of enzyme pools during purification was esrimdted using the Bradford 11976) assay (BioRad). The concentration of B sample of homogeneous threonine deaminase with an absorbance at 280 nm of 1.836 was determmed from a specific extinction coefficient of 0.74 em2 mg-1 derermined i"te~e,omet~ic~lly in a Beckman Model E ultracentrifuge using Rayleigh optics. The interference patterns were phot~graphe~ on Spectroscopic I1G plates (Kodak) and fringes were counted on a Nikon optical camparitor with a digital readout. A value of 4.1 fringes (mg/ml)-' (Babul and Stellewagen. 1969) was used to calculate an extinction coefficient for threonine deaminase in 0.05 M potassium phosphate, pH 7.5 with 0.1 mM EDTA. The value determined by this method was in good agreement with the protein concentration measured from amino acid analysis on the same sample.
&&ha& & J -Iktemrination of the pyridoxal phosphate content of purified threonine deaminase was performed by two methods. The first method relied on by U'ada and Snell (1961) with the modification suggested by Miles (1975 (Johnson and Fraser, 1985). The values for which the maximal slope was taken as nH, and the intercept on the abscissa taken as the K0.5 Vmax were then used to construct Hill plots, log (Vmar -V)/V ver~ur log [L-threonine] from (llill, 1910).

Clonine and Modification P f U f O r
Hieh-Level Exmession of Threonine Deaminase: In order to obtain gram quantities of threonine deaminase for biochemical and biophysical studles. a strategy was devised to modify ilvA. the structural gene for threonine deaminase, to facilitate subcloning the gene into different expression vectors for optimum expression. The firat step of this goal was to subclone the ilvA gene into pUCl18 (Vieira and Messing, 1987) for modification by site-directed mutagenesis. The ilvA gene was excised from pRWlB (Wek and Ilatfield. 1987) (kindly supplied by G.W. Hatfield. University of California, Irvine) by restriction digestion of plasmid DNA with BomH I and Bgl I1 to generate a 2.5 kb fragment.
agarose (FMC BioProducts). The ilvA containing fragment was then ligated into BomH I which was separated from unwanted DNA by electrophoresis in low gelling temperature dlgested pUC118 as described by Struhl erol. (1985) and the reaction mixture was used to transform Escherichia colr strain JV30 to ampicillin resistance.
to eliminate unnecessary sequences surrounding the ilvA gene to generate a "cassette" for rapid the first nucleotide at Arg466. Finally, an oligonucleotide, 5'-CGCCCGGGTAGAmAACCAACCCGCC-3'. was used to remove downstream sequences from the ilvY gene and, in addition, to remove a potential stemloop structure centered 21 bp downstream from the ilvA TAG stop codon. This oligonucleotide also changed the TAG stop codon to TAA. with the concomitant introduction of a unique Hind I11 restriction site. Thus, digestion of the the resulting plasmid. PEES, with the only the rlvA coding region. The ilvA "cassette" was then subcloned into similarly digested restriction enzymes Nco I and Hind 111 yields a 1545 bp fragment or "cassette" that contains pKK233-2 (Pharmacia-LKB Biotechnologies) to yield pEE6. mihis vector contains the hybrid rrp-lac (rrc) promoter (Amann and Brosius, 1985) with ilvA in a perfect, in frame fusion with the promoter and ribosome binding site to achieve expression of auhcntk enzyme.

Cell G r o w t h : Escherichia coli strain T31-4-452 (rhi-/, lrpEamY82Y, rrpAa,Y76/.
transformed to ampicillin resistance with pEE6 on LB agar plates containing 100 ug/ml ampicillin (Miller, 1972). Individual colonies were inoculated into liquid medium containing supplemented with 0.01% pyridoxine and 100 uglml ampicillin. At mid-log phase, 24 g yeast extract, 12 g tryptone, 2.3 g KHzPOI, 12.5 g KzHP04, 4 ml glycerol per liter, and cultures were grown an additional 18 hr prior to harvest. The amount of threonine deaminase isopropylthio-P-galactoside (IFTO) (BRL) was added to a final concentration of 1 mM and growth was continued to obtain a greater yield of cells and enzyme. High-level expression of reached a maximum level about 4-6 hr after induction, but since the cells continued to multiply, threonine deaminase was easily assessed by the intensely yellow color of the harvested cells, Cells have been stored up to six months at -8OOC with no apparent detrimental effect on threonine deaminase. passage of the suspension through a French Press cell at 8,WO to 9.000 psi. The extract was allowed to cool to 4% and then the French Press treatment was repeated. Unbroken cells and debris were removed by centrifugation for 30 min at 5,000 x g at 4OC in a Beckman 12-21 centrifuge to yield 170 ml of intensely yellow extract.
Ammonium sulfate was added dropwise to a stirring crude extract from a saturated solution at approximately pH 7.0 containing 50 mM potassium phosphate, 10 mM isoleucine. 1 mM EDTA and I mM pyridoxal phosphate to achieve 20% saturation (42.5 ml). After 1 hr at 40C. the slurry was centrifuged at 5,000 x g for 30 min. Ammonium sulfate solution was then added to the supernatant to reach 50% (114 ml). and after 1 hr the mixture was centrifuged to remove the precipitate. The intensely yellow precipitate containing threonine deaminase was dissolved in 50 mM potassium phosphate, 1 mM EDTA, 1 mM isoleucine, 1 mM dithiothreitol and 0.1 mM pyridoxal phosphate at pH 7.0 and dialyzed with several buffer changes at 40C.
Approximately 100 ml of the dialysate was then applied to a DEAE-cellulose (Whatman) column ( The dialyzed enzyme (about 10 ml) was clarified by centrifugation at 30,000 x g for 15 equilibrated in 50 mM postassium phosphate. 0.1 mM dithiothreitol and 0.1 mM EDTA at pH mi" and then applied to a column (2.5 x 120 cm) of Sephacryl S-3WHR (Pharmacia) 7.0, at a flow rate of 20 mlihr. The fractions containing pure threonine deaminase were then either filter sterilized and stored at 40C or dialyzed against 80% saturated ammonium sulfate homogeneous threonine deaminase that was capable of storage at 4OC at a concentration of (as described above) for long term storage. It is of interest to note that this protocol yielded dithiothreitol and 0.1 mM EDTA at pH 7.5 and as judged by constant kinetic parameters in approximately I mg/ml for at least two months in 50 mM potassium phosphate, 0.1 mM enzyme assays.
determination was removed from an ammonium sulfate slurry and dialyzed overnight against Threonine deaminase for use in steady-state kinetics or pyridoxal phosphate 50 mM potassium phosphate, pH 7.5, 0.1 mM EDTA, 0.1 mM dithiothreitol and 0.1 mM pyridoxal phosphate. The enzyme was then desalted by passage through a column ( I x 40 cm) of Sephacryl S300HR equilibrated in 50 mM potassium phosphate, pH 7.5. 0.1 mM EDTA. The fractions containing threonine deaminase were then dialyzed overnight against the equilibration buffer. These procedures were performed in the dark at 4oC.

RESULTS
Cloning and High-Level Expression of Threonine Deaminase . In order IO undertake structural gene for threonine deaminase. into plasmid vectors for mutagenesis of the gene and genetic and biochemical studies on threonine deaminase. it was desirable to clone ilvA. the expression of the enzyme. This could be readily accomplished since a portion of the ilv operon containing ilvA had been cloned from a Clarke-Carbon library into pUC8 (Wek and Hatfield. 1987). The strategy employed in these plasmid constructions, with a cartoon of the resulting mutagenesis plasmid, pEE5. and expression plasmid, pEE6, is presented in Fig. 1. These plasmids were constructed in four steps. First, an ilvA containing fragment was cut from pRWlB and ligated into pUC118. Second, three site directed mutagenesis experiments were performed to modify ilvA such that restriction enzyme sites were engineered at the start and stop codons of the structural gene. effectively generating a "cassette" containing only the ilvA coding sequence. Third, the '*cassette" was subcloned into pUCI20 to yield pEE5. which is useful for DNA sequencing and mutagenesis experiments. Fourth. the "cassette" was subcloned into pKK233-2 to yield pEE6, which places ilvA under control of the strong. inducible ITC promoter for enzyme expression. A determination of the DNA sequence of the ilvA gene was in excellent agreement with that reponed by Cox erol. (1987) and Lawther eral. (1987).  was performed as described under EXPERIMENTAL PROCEDURES to constmct an "casette" containing only the ilvA coding sequence. 7he ilvA "cassette" was then subcloned into pUCl20 yielding pEE5 for use in DNA sequencing and mutagenesis experiments or pKK233-2 yielding pEE6 for IPTG-inducible expression of threonine deaminase from the lrc promoter. E.colr strain T31-4-452. harboring a deletion in the chromosome that removes the wild-type ilvA gene, was grown to mid-log phase in rich medium. at which lime the culture was made I mM in IPTG. Cultures were grown another an extract from cells containing pEE6. which contains the ilvA gene expressed from the rrc 16-18 hr. harvested by centrifugation, and lysed by passage through a French press. Lane 3 is promoter. Lane 2 is an extract from cells containing an identical plasmid without the ilvA gene (pKK233-2). Threonine deaminase chains migrate with a molecular weight of 56.000. Lanes I and 4 contain low molecular weight standards from Bio-Rad. They include: phosphorylase b. 92.500. bovine serum albumin, 66.200; ovalbumin, 45.000. carbonic anhydrase, 31,000, soy bean trypsin inhibitor. 21.500. and lysozyme. 14.400. Samples containing about 4 ug protein in IS SDS and 1% 2-mercaptoethanol were heated to l W C for 3 min prior to electrophoresis on a Pharmacia Phasf system. several sources, including E. coli K-12. but only in small amounts. A detailed study of the Purification of Threonine Deaminase -Threonine deaminase has been purified from allosteric transition of this enzyme requires that large quantities of homogeneous enzyme be prepared with relative ease. The high-level expression of the enzyme permitted the development of rapid and efficient protocol for the preparation of gram quantities of enzyme.
The procedure involves selective ammonium sulfate precipitation. DEAE-cellulose chromatography. butyl-sepharose chromatography. and finally gel filtration on Sephacryl These steps yield large quantities of highly pure and highly active enzyme (about 8 mg purified S300HR. A summary of the yields at each step using this protocol is presented in Table 1. enzyme per g wet cell weight) in about a 50% overall yield. The purity of threonine deaminase prepared by this protocol was demonstrated by denaturing polyacrylamide gel electrophoresis as shown in Fig. 3. which shows a single band at approximately 56.000 daltons. Amino acid sequence analysis revealed that 95% of the polypeptide chains of threonine deaminase were blocked. Using and excess of protein. however. it was possible to determine the fint twelve amino terminal residues. which were A-D-X-N-P-L-X-G-A-P-E-G (X = undetermined). This sequence. with the exception of Ser4 and Ser8. is in excellent agreement with that expected from the ilvA nucleotide sequence. The purification procedure has been scaled successfully to process up to 170 g cells from a 12 I fermentation culture. with a yield of 1.25 g purified enzyme. Lane 2 contam 1.6 ug purifxd threonine deaminase treated as described in Figure 2. Lanes I and 3 contain the low molecular weight standards described in Figure 2. Electrophoresis was performed or. a Pharmacia Phast system. 50 mM potassium phosphate. pH 7.5 at 2WC. Although previous r e p n s have quoted final The final specific activity of h i s preparation is 210 units/mg/min a1 SU mM Inreonme m values for the specific activity of threonine deaminase as 210 unitslmgtmin (Calhoun cr 01.. 1973) or 230 unitslmghin (Koemer et 01.. 1975). the assay conditions employed by these workers were quite different with respect to buffer composition and temperature. In previous studies. threonine deaminase was assayed under conditions in which the enzyme has increased activity: 0.1 M potassium phosphate. pH 8.0. 0.1 M ammonium chloride at 370C. In order to compare more accurately threonine deaminase prepared in this study with those described prevtously. enzyme assays were performed under conditions of buffer and temperature that conform exactly to those used in previous work. As can be seen in Fig. 4. steady-state kinetics performed in 0.1 M potassium phosphate. pH 8.0.0.1 M ammonium chloride. at 370C display a maximum specific activity of 630 unitslmglmin. or roughly three-times that previously reponed (Calhoun et al.. 1973: Koemer et al., 1975. Moreover, the enzyme displays a sigmoidal dependence of initial velocity on threonine concentration. This is especially apparent in Fig. 4(B), an Eadie plot which is concave downward, indicative of positive homotropic cooperativity (Hensley er 01.. 1981). A comparison of the steady-state kinetic parameters for threonine deaminase under different temperature and buffer Conditions is summarized in Table  II