©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
4-Chlorothreonine Is Substrate, Mechanistic Probe, and Mechanism-based Inactivator of Serine Hydroxymethyltransferase (*)

(Received for publication, January 31, 1995; and in revised form, April 10, 1995)

Heather K. Webb , Rowena G. Matthews (§)

From the Biophysics Research Division and Department of Biological Chemistry, the University of Michigan, Ann Arbor, Michigan 48109

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Serine hydroxymethyltransferase catalyzes the cleavage of a variety of -hydroxy-L-amino acids to form glycine and aldehyde products. 4-chloro-L-threonine has been synthesized and shown to be both a substrate and a mechanism-based inactivator of serine hydroxymethyltransferase. k values for the formation of glycine in the absence of tetrahydrofolate were determined for 4-chloro-L-threonine and other -hydroxyamino acid substrates; an inverse relationship between the rate of cleavage of the amino acid and the electrophilicity of the product aldehyde was demonstrated. 4-Chloro-L-threonine inactivates serine hydroxymethyltransferase in a time- and concentration-dependent manner and exhibits saturation of the rate of inactivation at high concentrations. Our evidence suggests that 4-chlorothreonine undergoes aldol cleavage, and generation of chloroacetaldehyde at the active site of the enzyme results in inactivation. Serine or glycine protect the enzyme against inactivation by chlorothreonine, while tetrahydrofolate does not. The enzyme is also protected from inactivation by 2-mercaptoethanol or by alcohol dehydrogenase and NADH. These studies suggest that halothreonine derivatives that generate electrophilic aldehyde products will be effective inhibitors of serine hydroxymethyltransferase and might be potentially useful chemotherapeutic agents.


INTRODUCTION

The pyridoxal-5`-phosphate-dependent enzyme, serine hydroxymethyltransferase (EC 2.1.2.1) catalyzes the reversible interconversion of serine and glycine (Reaction 1).


Reaction I

The enzyme charges Hfolate()with a one-carbon unit that is used by enzymes involved in the de novo synthesis of deoxythymidylate and purines and in the methylation of homocysteine to form methionine. Because deoxythymidylate and purines are required for DNA synthesis, and thus for cell division, serine hydroxymethyltransferase activity may limit the growth rate of cells. Several studies demonstrating higher serine hydroxymethyltransferase activity in hepatoma and myeloma cells than in untransformed cells indicate that the enzyme may be an appropriate target for anti-cancer chemotherapeutic agents, since rapidly dividing tumor cells rely more heavily on the de novo biosynthesis of DNA and hence require more serine hydroxymethyltransferase activity than slowly dividing cells(1, 2) . Therefore, specific inhibitors of serine hydroxymethyltransferase may be useful in cancer chemotherapy.

In addition to catalyzing the transfer of a hydroxymethyl group from serine to Hfolate, serine hydroxymethyltransferase also reacts with -hydroxyamino acids in the absence of Hfolate, converting them to aldehydes and glycine(3, 4) . Of these -hydroxyamino acid substrates, only L-serine and -methyl-L-serine, which are the slowest reactants in the absence of Hfolate, require Hfolate as a cosubstrate for efficient catalysis(5, 6) . The Hfolate-independent cleavage of L-serine to form glycine and formaldehyde is more than 10-fold slower than the reaction in the presence of Hfolate(6) . For substrates such as threonine and -phenylserine that do not require Hfolate for efficient catalysis, the reaction is thought to proceed by a retroaldol cleavage, and the accepted mechanism is shown in Fig. 1(7) . An important mechanistic question is whether the cleavage of the physiological substrate, serine, also involves retroaldol cleavage.


Figure 1: A kinetic mechanism for cleavage of -hydroxyamino acids in the absence of Hfolate. In step 1, the -hydroxyamino acid binds the internal aldimine form of the enzyme (EIA), and the external aldimine is formed by transimination (EEA). In step 2, C-C bond cleavage takes place to form a resonance-stabilized quinonoid form of the glycine carbanion (Equin), and the aldehyde product is released. In step 3, the quinonoid form of the glycine aldimine is protonated (Egly). In step 4, transimination takes place to release glycine and regenerate the internal aldimine form of the enzyme.



In this work we describe the synthesis of a serine analog, 4-chloro-L-threonine, and characterization of its properties as a substrate and inhibitor for serine hydroxymethyltransferase. We show that chlorothreonine is a substrate for serine hydroxymethyltransferase and that it is cleaved to form chloroacetaldehyde and glycine (Fig. 2), and we provide evidence that the chloroacetaldehyde generated at the active site is responsible for enzyme inactivation. The rate of cleavage of chlorothreonine is intermediate between the rates of cleavage of serine and threonine. Ulevitch and Kallen (8) previously demonstrated a relationship between the rate of cleavage of substituted -phenylserine substrates and the electrophilicity of the product aldehydes. Our work extends this relationship to include threonine, chlorothreonine, and serine, establishing that the mode of cleavage of all -hydroxyamino acid substrates, whether natural or unnatural substrates, is likely to be the same. While Hfolate accelerates the rate of cleavage of serine more than 200,000-fold, it accelerates the rate of cleavage of 4-chloro-L-threonine less than 4-fold. These studies suggest that halothreonine derivatives that are cleaved to produce highly electrophilic aldehyde products will be effective inhibitors of serine hydroxymethyltransferase that may be useful as chemotherapeutic agents.


Figure 2: The proposed mode of reaction of serine hydroxymethyltransferase with 4-chloro-L-threonine. A base-catalyzed retroaldol cleavage of the substrate results in release of chloroacetaldehyde product and formation of a quinonoid glycine aldimine. E B is an amino acid side chain that serves as the general base in this reaction.




EXPERIMENTAL PROCEDURES

Materials

Benzyl-(2S)-2-{[(benzyloxy)-carbonyl]amino} butanoic acid (or N-Cbz-L-glutamic acid--benzyl ester) was purchased from Bachem, Inc. Glucose-6-phosphate dehydrogenase and alcohol dehydrogenase were purchased from Sigma. Other chemicals used in the synthetic procedures were reagent grade or higher quality and were obtained from Aldrich or Sigma. The following buffers were used: Buffer A (50 mM HEPES, pH 7.5), Buffer B (50 mM potassium phosphate, pH 7.2, 0.1 mM pyridoxal-5`-phosphate, 0.3 mM EDTA and 2.0 mM 2-mercaptoethanol), Buffer C (100 mM sodium acetate, pH 5.1), Buffer D (70% acetonitrile, 0.10 M sodium acetate, pH 4.4), Buffer E (50 mM HEPES, pH 7.5, 25 mM NaSO, 1 mM EDTA), and Buffer F (0.10 M potassium phosphate, pH 7.0).

General Methods

H and C NMR spectra were recorded on a Bruker AM 360 (Fourier transform) or Bruker AM 300 (Fourier transform) instrument as indicated. Chemical shifts were referenced to the residual HOD resonance at 4.63 ppm (H NMR), a tetramethyl silane resonance at 0.00 ppm (H NMR), or the central peak of CDCl at 77 ppm (C NMR). Mass spectra were recorded on a 7070E J G analytical organic mass spectrometer. Elemental analyses were performed by Atlantic Microlabs, Inc., and by the University of Michigan Department of Chemistry Elemental Analysis Facility. Optical rotation was measured on a Perkin Elmer 241 Polarimeter. Protein concentrations were determined using the Bio-Rad protein assay(9) . Enzyme assays were performed on a Beckman DU Spectrophotometer outfitted with modern electronics and a recorder. All enzyme incubations and assays were performed at 30 °C in Buffer A unless otherwise noted.

Stereoselective Synthesis of 4-Chloro-L-threonine from N-Cbz-L-glutamate--benzyl ester(2

The synthesis of 4-chloro-L-threonine from N-Cbz-L-glutamic acid--benzyl ester (2) is shown in Fig. 3, and a detailed description of the synthesis is presented in the Ph.D. dissertation of Webb(10) . Benzyl (2S)-2-{[(benzyloxy)carbonyl]amino}-3-butanoic acid (3) was prepared by the method described by Hanessian and Sahoo(11) , and benzyl (2S,3R)-2-{[(benzyloxy)-carbonyl]amino}-3,4-epoxybutanoic acid (4) was prepared by a method similar to that described by Shaw et al.(12) . 16.7 g (48 mmol) of 50% m-chloroperoxybenzoic acid in CHCl was prepared as described in (13) . This solution was then transferred into a flask containing 3.28 g (9.6 mmol) of 3 and stirred for 120 h at 25 °C under nitrogen. The reaction was cooled on ice and extracted with 10% NaSO and then with Buffer F. The CHCl layer was dried with MgSO, and the solvent was removed under vacuum. The product was crystallized from an 8.5:1.5 mixture of hexane:ethyl acetate.


Figure 3: Outline of the synthesis of 4-chloro-L-threonine.



Benzyl (2S,3R)-2-{[(benzyloxy)carbonyl]amino-3-hydroxy-4-chlorobutanoic acid (5) was prepared by a regiospecific ring opening of the epoxide (4). 0.12 g (0.32 mmol) of the protected epoxide (4) was placed in a dried round bottom flask. 7.0 ml (2 eq) of 0.1 M LiCuCl in dry tetrahydrofuran was added to the flask, and the reaction was stirred at room temperature for 18 h. The reaction was neutralized with 25 ml of Buffer F and then extracted with diethyl ether. The diethyl ether layer was dried with MgSO and was removed under vacuum. The product was crystallized from a 4:1 mixture of hexane:ethyl acetate.

(2S,3R)-2-Amino-4-chloro-3-hydroxy-butanoic acid (4-chloro-Lthreonine) (1) resulted from the deprotection of 5. 0.1 g (0.26 mmol) of the protected chlorohydrin (5) was suspended in 15 ml of 6 N HCl and heated to 80 °C for 8 h. The solution was cooled and extracted with diethyl ether, and the product was isolated by lyophilization of the aqueous layer. The yield was 40 mg (94%) and decomposed at 140-145 °C. A single peak was observed for 4-chloro-L-threonine by ion exchange amino acid analysis and postcolumn derivatization with ninhydrin. The retention time of 4-chloro-L-threonine was 56 min, and that of threonine was 59 min.

(2S, 3R)-2-Amino-3-hydroxybutanoic acid (L-threonine) (7) was prepared from 6 to determine the stereochemistry of 5. Benzyl (2S,3R)-2-{[(benzyloxy)-carbonyl]amino}-3-hydroxy-4-iodobutanoic acid (6) was prepared from 5 by halide exchange using the Finkelstein reaction(14) . The product 6 was dissolved in 15 ml of methanol, and 30 mg of 10% Pd on carbon was added. The suspension was placed in a Parr hydrogenator at 45 p.s.i.g. H and shaken for 1 h at room temperature. The suspension was then filtered through Celite, and the methanol was removed under vacuum. [] (c = 1, HO) reported for 7 was -27° and observed was -25°(15) .

Identification of the Breakdown Product of 4-Chlorothreonine

Incubation of 4-chloro-L-threonine in Buffer A or incubation of chloroacetaldehyde and [U-C]-glycine with buffered enzyme results in formation of an amine breakdown product from 4-chloro-L-threonine with an approximate half-time of 12 h. Mass spectroscopy studies demonstrated that 4-chloro-L-threonine had lost its chlorine. The NMR spectrum was similar to that of threonine, except there were only two protons and there was a down-field shift for the and protons. These results are consistent with cyclization of 4-chloro-L-threonine with the elimination of chlorine to produce 2-amino-3-hydroxy-5-lactone. During incubation in Buffer A, the half-time for breakdown of 4-chloro-L-threonine to form the lactone is approximately 12 h, and no glycine formation is detected.

Enzyme Purification and Assays

The cytosolic isoenzyme of serine hydroxymethyltransferase was purified from pig liver using the method described in Matthews et al.(16) and was given to us by Professor Leodis Davis. All incubations and activity assays were performed after exchange of the enzyme into Buffer A. A unit of enzyme activity is defined as 1 µmol benzaldehyde formed min from 100 mMDL-threo--phenylserine in Buffer E (17) ; the formation of benzaldehyde was monitored at 279 nm. A second assay method was developed based on amino acid sequencing methods and involves conversion of the substrate and amino acid products in the assay mixture to phenylthiohydantoin derivatives and separation of the derivatized amino acids by reversed-phase HPLC. Samples containing 20 µg of amino acid were removed at timed intervals from an incubation mixture of enzyme and -hydroxyamino acid substrate in Buffer A. The enzyme in the sample was denatured at 95 °C, and the sample was dried using a Speed-Vac vacuum pump. The amino acid samples and standards were then derivatized with phenylisothiocyanate as described by Shively(18) . The samples were dissolved in 1.0 ml of Buffer C, and 80 µl portions were injected onto an HPLC with a 4.6 mm 25 cm Beckman Ultrasphere ODS 5-µm column. Products were detected by absorption at 254 nm. The phenylthiohydantoin derivatives of the amino acids were separated isocratically with 90% Buffer C and 10% Buffer D at a flow rate of 1 ml/min. The retention time of glycine phenylthiohydantoin is 9.3 min. Glycine concentrations ranging from 10 pmol to greater than 150 pmol gave a linear response, and the amount of glycine formed was calculated by comparison with a standard curve. No significant loss in recovery was observed due to the presence of enzyme at the levels used in these assays. The retention time for the phenylthiohydantoin derivative of 4-chloro-L-threonine is 10.5 min, but when 4-chloro-L-threonine is heated to 95 °C and derivatized in buffer it decomposes and elutes as three peaks at 8.6, 13.6, and 15 min. Measurements of -hydroxyamino acid cleavage in the presence of Hfolate were performed under argon in Buffer A with 0.025 M -mercaptoethanol.

Inactivation Assays

4-Chloro-L-threonine was prepared and stored as the hydrochloride salt. Prior to use for enzyme inactivation, 4-chloro-L-threonine was dissolved in 0.05 M HEPES and 0.1 M NaOH, yielding a final solution of 0.1 M 4-chloro-L-threonine, 0.1 M NaCl, 0.05 M HEPES, pH 7.5. The concentration of chloride ion in the inactivation reactions, 1-10 mM, has little effect on enzyme activity measured at pH 7.5. The percentage of residual enzyme activity was determined by comparison with the initial activity in the same reaction. The enzyme was prepared for inactivation studies by exchanging into Buffer A. The inactivation incubation mixtures contained enzyme and freshly diluted 4-chloro-L-threonine at varying concentrations. Samples were taken at timed intervals and tested for activity using the spectrophotometric and the HPLC assays. The inactivation assay performed in the presence of alcohol dehydrogenase contained 2 µM enzyme active sites, 10 mM 4-chlorothreonine, 50 mM HEPES, pH 7.5, 15 µg of alcohol dehydrogenase, 0.4 mM NADH, 7.5 units of glucose-6-phosphate dehydrogenase, and 3.3 mM glucose-6-phosphate in a total volume of 150 µl.


RESULTS

Synthesis of 4-Chloro-L-threonine

The stereoselective synthesis of 4-chloro-L-threonine from the -benzyl ester of N-Cbz-L-glutamic acid (2) is shown in Fig. 3. Parts of the synthesis described in this paper draw on the work of the groups of Hanessian (11) and Rapoport(12) . This route begins with oxidative decarboxylation of protected glutamic acid 2, resulting in the vinyl glycine derivative (3). The vinyl glycine derivative 3 is then converted in high yield to the epoxide 4 using m-chloroperoxybenzoic acid. Unlike previous work with the -methyl ester(12) , which yielded a 4:1 ratio of the threo to erythro diastereomers, the epoxidation of the benzyl ester-protected amino acid results in the formation of >95% of the threo diastereomer; the erythro form is undetectable by either C or H NMR. Using the general method described by Bell and co-workers(19) , the epoxide ring (4) was then regiospecifically opened using LiCuCl to form the protected chlorohydrin (5). The benzyl-protected chlorohydrin was deprotected by acid hydrolysis to form the stable hydrochloride salt.

4-Chlorothreonine Is a Substrate

The steady state kinetics for several -hydroxyamino acid substrates were determined in the absence of Hfolate using the same enzyme source, buffer, and temperature (Table 1). The rate of glycine formation was measured for each substrate tested, so that different assays for different substrates were avoided and a direct comparison of k values is possible. For substrates that had previously been studied, the values obtained in our studies compare well with values we calculated from published data, despite the fact that earlier studies employed enzyme from different sources and used different assays and conditions(8, 17, 20) . Kvalues for the amino acid substrates decrease as the k values decrease, as noted previously(8) .



4-Chloro-L-threonine serves as a substrate for serine hydroxymethyltransferase with a k value that is intermediate between serine and threonine. Glycine production was observed by HPLC analysis of the amine products in the incubation mixture before and after incubation of 4-chloro-L-threonine with serine hydroxymethyltransferase. A k value of 0.033 ± 0.001 s and a K value of 1.6 ± 0.7 mM were determined for 4-chloro-L-threonine (Table 1). The production of glycine from 4-chloro-L-threonine by the enzyme was corroborated by HPLC and thin layer chromatographic analysis of the mixture before and after incubation with the enzyme.

Hfolate Increases the Rate of 4-Chlorothreonine Cleavage

The rates of turnover of L-serine and 4-chloro-L-threonine were measured at varying concentrations of Hfolate with resulting values for k of 21 ± 5 s and 0.19 ± 0.03 s, respectively. These measurements were performed under argon in the presence of 25 mM -mercaptoethanol to minimize the oxidation of Hfolate and enzyme inactivation by the chloroacetaldehyde product. The presence of this concentration of -mercaptoethanol does not significantly affect k for serine cleavage in the absence of Hfolate but changes the observed k value for 4-chloro-L-threonine cleavage from 0.033 to 0.046 s. As previously observed(20) , k for serine was more than 5 orders of magnitude greater in the presence of Hfolate than in its absence, with a k of 21 ± 5 s with Hfolate as compared with 0.0001 s without Hfolate. The K for (6R,S) Hfolate with serine as the second substrate is 350 µM. The rate constant for 4-chloro-L-threonine turnover, on the other hand, shows only a 4.1-fold increase in the presence of Hfolate with a k value of 0.19 s and a K value for Hfolate of 87 µM (Fig. 4). This effect of Hfolate is not due to protection of the enzyme against inactivation by chloroacetaldehyde, which occurs very slowly compared with turnover.


Figure 4: Stimulation of the rate of 4-chloro-L-threonine cleavage by Hfolate. The solidcircles show observed rate constants for the cleavage of 4-chlorothreonine as a function of the concentration of Hfolate. Each point represents a k value obtained from a Michaelis-Menten plot of data obtained at a series of concentrations of 4-chlorothreonine at the indicated concentration of Hfolate. In the presence of saturating Hfolate, k is 0.19 s, which is a 4.1-fold increase over the k for 4-chloro-L-threonine measured under the same conditions in the absence of Hfolate.



4-Chloro-L-threonine Inactivates Serine Hydroxymethyltransferase

Criteria for mechanism-based inhibitors (21) include (a) inactivation in a time- and concentration-dependent manner that saturates at high concentrations of the inhibitor, (b) protection against enzyme inactivation by the physiological substrate, (c) inactivation at a rate no faster than the rate of substrate consumption, (d) irreversible covalent bond formation, with no return of activity after removal of the inhibitor, and (e) no release of an activated species prior to enzyme inactivation.

4-Chloro-L-threonine inactivates serine hydroxymethyltransferase in both a time- and concentration-dependent manner (Fig. 5). The inactivation reactions are sufficiently slow relative to the rate of catalysis of aldol cleavage that equilibrium is reached early in the inactivation progress, particularly at concentrations of chlorothreonine below 10 mM. The equilibrium constant for formation of chloroacetaldehyde and glycine from chlorothreonine is 5 10M, as determined by measurement of the amount of glycine formed from chlorothreonine at equilibrium. The rate of inactivation saturates at high concentrations of 4-chloro-L-threonine, and the apparent K of 1.7 ± 0.5 mM is consistent with the formation of a Michaelis complex between the enzyme and 4-chloro-L-threonine. At saturating levels of chlorothreonine, k was found to be (3.0 ± 0.2) 10 s. This rate of inactivation is significantly slower than the rate of glycine formation from 4-chloro-L-threonine of 3.3 10 s. The partition ratio was calculated from the ratio of k to k and found to be 940; at different concentrations of chlorothreonine the same partition ratio is observed.


Figure 5: A, the inactivation of serine hydroxymethyltransferase is time- and concentration-dependent. Rates of inactivation of the enzyme were measured in the absence of 4-chloro-L-threonine (circles) and in the presence of 2.5 (diamonds), 6 (triangles) and 10 (squares) mM initial concentrations of 4-chloro-L-threonine. B, a plot of the apparent rate of inactivation as a function of 4-chlorothreonine concentration. Because enzyme turnover rapidly results in equilibration of chlorothreonine with glycine and chloroacetaldehyde, the equilibrium concentrations of chlorothreonine, rather than the initial concentrations, are plotted. The values for K of 1.7 ± 0.5 mM and k of (3.0 ± 0.2) 10 s were calculated as described by Kitz et al.(24) using a nonlinear fit to the data and the KaleidaGraph plot fitting program (Synergy Software, Reading, PA).



The enzyme is apparently permanently inactivated by 4-chlorothreonine. There is no observable return of activity when 4-chloro-L-threonine is removed by either gel filtration or ultrafiltration. Incubation of completely inactivated enzyme with pyridoxal-5`-phosphate and -mercaptoethanol after the removal of 4-chloro-L-threonine does not result in a return of enzyme activity. These results indicate that loss of cofactor is not the mode of inactivation. There is no discernible change in the apparent subunit molecular weight as determined by SDS-polyacrylamide gel electrophoresis; thus gross modification of the enzyme due to covalent cross-linking of subunits does not occur.

Both serine and glycine protect against inactivation by 4-chloro-L-threonine (data not shown). The enzyme incubated with 5 mML-serine (a concentration significantly above its K) and 5 mM 4-chloro-L-threonine (near its K) showed no detectable loss of activity over 400 min. Glycine also protects against inactivation, and this protection is maximal at glycine concentrations above 1 mM. When the enzyme is incubated with 10 mM 4-chloro-L-threonine and 10 mM glycine, the rate of inactivation is 46% of that seen for incubations with 4-chloro-L-threonine alone. These results are consistent with 4-chloro-L-threonine competing with serine and glycine for binding to the active site. In contrast, Hfolate did not protect serine hydroxymethyltransferase against inactivation by 4-chloro-L-threonine but instead accelerated the rate of inactivation (data not shown).

-Mercaptoethanol and alcohol dehydrogenase also protect serine hydroxymethyltransferase against inactivation by 4-chloro-L-threonine. Enzyme incubated with 10 mM 4-chloro-L-threonine and 10 mM -mercaptoethanol shows no significant loss of activity over 7 h. There is no detectable modification of 4-chloro-L-threonine by -mercaptoethanol as observed by thin layer chromatographic analysis. The presence of alcohol dehydrogenase and NADH also prevents inactivation of the enzyme by 4-chloro-L-threonine. The predicted product, chloroacetaldehyde, is known to be reduced by alcohol dehydrogenase (22) .

The protection by alcohol dehydrogenase and -mercaptoethanol might indicate that chloroacetaldehyde leaves the active site before inactivation, since a large enzyme has access to the product. Indeed, chloroacetaldehyde has been shown to inactivate serine hydroxymethyltransferase(22) , and we have determined that the apparent rate of inactivation is first order in chloroacetaldehyde between 0 and 10 mM concentrations. However, no lag phases are seen in the inactivation progress experiments shown in Fig. 5A, and in fact points on each of the lines actually suggest a burst phase early in the inactivation process. Such observations are only consistent with the assumption that inactivation occurs before chloroacetaldhyde leaves the active site of the enzyme, with the burst phase of inactivation being associated with the change in concentration of chlorothreonine as equilibrium is approached. It is possible that the active site of serine hydroxymethyltransferase is accessible to a large protein. Studies of Stover et al.(23) have established that native serine hydroxymethyltransferase converts from an open to a closed form on formation of the external aldimine, but it is not known whether the binding of substituted -hydroxyamino acids also results in closure of the active site.


DISCUSSION

The data obtained in this study document a linear free energy relationship between the rate of cleavage of -hydroxyamino acid substrates by serine hydroxymethyltransferase in the absence of Hfolate and the hydration equilibria of the product aldehydes, thus demonstrating that cleavage of these substrates is accelerated by electron-donating substituents on the -carbon. The rate-limiting step in cleavage of -phenylserine substrates in the absence of Hfolate is associated with C-C bond cleavage and aldehyde release (Fig 1, step2), and this study establishes that the same step is rate-limiting for all -hydroxyamino acid substrates tested. Such information is useful in designing active site-directed inhibitors for serine hydroxymethyltransferase, and one inhibitor, 4-chloro-L-threonine, has been characterized in this study.

There Is a Linear Free Energy Relationship between the Rate Constants for Cleavage of -Hydroxyamino Acids and the Hydration of the Product Aldehydes

Ulevitch and Kallen studied the rates of cleavage of substituted -phenyl serine substrates of serine hydroxymethyltransferase(8) . They demonstrated a linear relationship between the logarithm of k for cleavage and the Hammett value for substituent, which indicated that the rate of cleavage of substituted -phenylserines is accelerated by electron-donating substituents on the phenyl ring. These nonphysiological substrates are cleaved rapidly by serine hydroxymethyltransferase in the absence of Hfolate. Our examination of the k values for cleavage of nonaromatic -hydroxyamino acid substrates by serine hydroxymethyltransferase suggested a correlation between these values and the electrophilicity of the product aldehydes formed, a correlation that extends from the substituted -phenylserines to include threonine, chlorothreonine and the physiological substrate, serine. The logarithms of k values for -hydroxyamino acids cleaved in the absence of Hfolate were found to be linearly related to the pK values for hydration of the aldehyde product over 5 orders of magnitude (Fig. 6). A simple linear free energy relationship implies that a single transition state dictates the rate for each of the reactions obeying the relationship. The strong linear relationship seen in Fig. 6is indicative of a common rate-determining step for all of the substrates tested, from serine to the -phenylserines. Furthermore, the slope of 0.93 indicates that the -substituent has a large effect on the rate-determining step.


Figure 6: Linear free energy plot of log k for -hydroxyamino acid cleavage in the absence of Hfolate versus the pK of the product aldehyde. The k values are those listed in Table 1. From left to right the substrates are serine, 4-chlorothreonine, threonine and allo-threonine, p-nitro-erythro--phenyl serine, 3,4-dichloro-erythro-phenylserine, m-chloro-erythro--phenylserine, p-chloro-erythro--phenylserine, erythro--phenylserine and threo--phenylserine, and p-methoxy-erythro--phenylserine. Opencircles indicate values for k calculated from work by others (references given in Table 1), while solidcircles are shown for data measured in this study. When there is more than one value listed for a single compound, the k determined in this study was used. The K values are from the following sources: formaldehyde(25) ; chloroacetaldehyde(26) ; acetaldehyde(27) ; benzaldehyde(28) ; and p-nitrobenzaldehyde, p-chlorobenzaldehyde, m-chlorobenzaldehyde, and p-methoxybenzaldehyde(29) .



What Is the Rate-limiting Step?

Fig. 1describes a kinetic mechanism that delineates the major steps in turnover of a generic -hydroxyamino acid in the absence of Hfolate(8) . Steps 1, 3, and 4 from Fig. 1can be eliminated as possible rate-limiting steps. Kinetic studies of the condensation of glycine and formaldehyde (aldol condensation to form serine) were performed by Chen and Schirch (20) , indicating that the reaction was ordered, with glycine binding preceding that of formaldehyde. Thus, steps 3 and 4 in the forward direction would not be expected to show any substituent effect, since they would occur after release of the product aldehyde. The rate-determining step is also not the binding step, step 1, since stopped-flow experiments have shown that formation of the external aldimine occurs in the instrument dead time with -phenylserine(8) . The enzyme-serine aldimine is also completely formed before measurements can be made in a recording spectrophotometer at 10 °C, which indicates a rate of formation of the serine aldimine that is much faster than k for the reaction with serine (6) . These two substrates, -phenylserine and serine, exhibit two extremes in rate, and neither rate is limited by substrate binding. Thus, steps 1, 3, and 4 are not the rate-limiting steps in cleavage of -hydroxyamino acids. This leaves step 2 as the rate-limiting step as described by Ulevitch and Kallen(8) .

What Can We Infer about the Mechanism of -Hydroxyamino Acid Cleavage in the Absence of Hfolate?

From the free energy relationship shown in Fig. 6, we can conclude that the rate of cleavage increases as the substituent on -hydroxyamino acid substrates becomes more electron-donating, indicating either an increase in positive charge or a decrease in negative charge as the reaction approaches the transition state. Based on their measurements with substituted -phenylserines, Ulevitch and Kallen (8) interpreted this observation as indicating a rapid removal of the proton from the -hydroxy group, followed by a rate-limiting C-C bond cleavage (Fig. 7). Approach to the transition state for this base-catalyzed aldol cleavage would involve a decrease in negative charge. The strong correlation between rate (k) and hydration equilibrium of the product suggests that the transition state for this reaction is very late and resembles the aldehyde, with very little residual negative charge on the carbonyl, so that the reaction is disfavored by electron-withdrawing substituents that stabilize the alkoxide. Hfolate, which accelerates the reaction with the slowest substrates, must either alter the reaction mechanism or selectively destabilize the alkoxide or stabilize the nascent aldehyde during aldol cleavage.


Figure 7: Mechanism proposed by Ulevitch and Kallen (8) for general base-catalyzed aldol cleavage of -phenylserine substrates in the absence of Hfolate. Deprotonation of the -hydroxyl group was assumed to occur in a rapid step prior to the rate-limiting C-C bond cleavage.



According to this hypothesis, -hydroxyamino acid substrates with electron-withdrawing substituents that give rise to highly electrophilic aldehydes will be very effective reversible inhibitors of serine hydroxymethyltransferase, because they will react at extremely slow rates and will thus block the reaction of the enzyme with serine and/or glycine. 4-Chloro-L-threonine represents a synthetically accessible example of this type of potential inhibitor, but 4-fluorothreonine and 4-trifluorothreonine would be expected to be even more potent inhibitors. Clearly, the efficacy of halothreonine substrates as inhibitors would be compromised if their rate of cleavage was substantially accelerated in the presence of Hfolate. However, in contrast to serine, which is cleaved more than 200,000-fold faster in the presence of Hfolate than in its absence ( Table 1and (20) ), Hfolate only accelerates the rate of cleavage of 4-chloro-L-threonine 4-fold (Fig. 4). Thus, even in the presence of Hfolate, halothreonine inhibitors may be expected to retain their potency.

Mechanism of Inactivation

The observed kinetics of inactivation of serine hydroxymethyltransferase are consistent with conversion of 4-chlorothreonine to an active site-directed affinity label, chloroacetaldehyde. This inactivation probably occurs as a result of partitioning of chloroacetaldehyde formed at the active site between release and alkylation of the protein. 4-Chloro-L-threonine is probably unsuitable for chemotherapy, because it leads to the release of a reactive electrophile into solution following reaction with serine hydroxymethyltransferase. However, fluorinated derivatives such as 4-trifluoro-L-threonine or the corresponding allo-threonine derivative would be expected to react extremely slowly with serine hydroxymethyltransferase and might thus be suitable for chemotherapy.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant R37 GM24908 from NIGMS. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Biophysics Research Division, 4024 Chemistry Bldg., 930 N. University, Ann Arbor, MI 49109-1055. Tel.: 313-764-9459; Fax: 313-764-3323; Rowena.G.Matthews{at}um.cc.umich.edu

The abbreviations used are: Hfolate, tetrahydrofolate; CH-Hfolate, N,N-methylenetetrahydrofolate; HPLC, high performance liquid chromatography; Cbz-, benzyloxycarbonyl-.


ACKNOWLEDGEMENTS

We thank Professor James K. Coward for the use of equipment and space in his laboratory and for the helpful advice he and his students John R. Lakanen and Mark R. Burns provided. We thank James T. Drummond for helpful discussions and Professor Leodis Davis for the gift of serine hydroxymethyl transferase.


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