THE OF CHEMISTRY Val. 266, 3, of 25, pp. Q 1991 by for and … · 2001-06-07 ·...

THE JOURNAL OF BIOLOGICAL CHEMISTRY Q 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Val. 266, No. 3, Issue of January 25, pp. 1543-1550,1991 Printed in U. S. A.

5-Formyltetrahydrofolate Polyglutamates Are Slow Tight Binding Inhibitors of Serine Hydroxymethyltransferase*

(Received for publication, June 27, 1990)

Patrick Stover and Verne Schirchg From the Department of Biochemistry and Molecular Biophysics, Virginia Commonwealth Uniuersity, Medical College of Virginia, Richmond, Virginia 23298

The interaction of the mono- and triglutamate forms of 5-methyltetrahydrofolate and 5-formyltetrahydrofolate with serine hydroxymethyltransferase were determined by several methods. These methods included determining dissociation constants by observing the absorbance at 502 nm of a ternary complex of the enzyme, glycine, and the folate compounds; determining inhibition constants from steady-state reactions; and determining the rate of formation and breakdown of the enzyme inhibitor complex by rapid reaction kinetics. Studies of the dissociation and inhibitor constants showed that both 5-methyltetrahydrofolate and 5-formyltetrahydrofolate have essentially the same affinity for the enzyme-glycine binary complex. How- ever, rapid reaction and steady-state kinetic studies showed that the triglutamate form of 5-formyltetrahydrofolate both binds and is released much more slowly from the enzyme-glycine binary complex, compared with the triglutamate form of 5-methyltetrahydrofolate. The results also showed that only one rotamer of 5-formyltetrahydrofolate binds at the active site of serine hydroxymethyltransferase. The results are discussed in terms of the possible role of 5-formyltetrahydrofolate polyglutamates in regulation of one- carbon metabolism.

The conversion of serine and H,PteGlu‘ to glycine and 5,lO-CH2-H,PteGlu by serine hydroxymethyltransferase (SHMT) provides the cell with approximately 70% of the C1 units required for the synthesis of thymidine, purines, choline, and methionine. SHMT, which exists at the branch point of one carbon metabolism, binds all reduced folate coenzymes in the cell with high affinity, with the exception of 10-CHO- H4PteGlu. The determination of intracellular folate pools by Horne et al. (1989) revealed that over 50% of folate derivatives in rat liver are N5-substituted in the form of 5-CH3-H4PteGlun

* This work was supported in part by Grant GM 28143 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. + To whom correspondence should he addressed.

The abbreviations used are: H,PteGlu, tetrahydropteroate; cSHMT, rabbit liver cytosolic serine hydroxymethyltransferase; mSHMT, mitochondrial serine hydroxymethyltransferase; C,-THF synthase, C,-tetrahydrofolate synthase; 5,lO-CH’THF synthetase, 5,lO-methenyltetrahydrofolate synthetase; 5-CH3-H4PteGlu,, (6s)- 5-methyltetrahydropteroate containing n glutamate residues; 5-CHO- H,PteGlu,,, (GS)-5-formyltetrahydropteroate containing n glutamate residues; N”-H,PteGlu,, both (GS)-5-methyl- and ( 6 s ) 5-formyltetrahydropteroate; 10-CHO-H4PteGlu,, ( 6 R ) 10-formyltetrahydropteroate containing n number of glutamate residues; KMES, potassium 2-[N-morpholino]ethanesulfonate.

(40%) and 5-CHO-HrPteGlun (10%). Both of these N5-substituted folates are effective inhibitors of SHMT, suggesting that they may be important in regulating the major entry point of C, units (Schirch and Ropp, 1966; Matthews et al., 1982).

5-CH3-H4PteGlu is formed by the reduction of 5,10-CH2- H4PteGlu by the flavoenzyme, methylenetetrahydrofolate re- ductase. The enzyme is strongly (Ki = 3 p M ) but slowly inhibited by S-adenosylmethionine. Jencks and Matthews (1987) further characterized this inhibition and identified S- adenosylmethionine as a slow binding allosteric inhibitor of the enzyme. Regulation of this enzyme is critical to one- carbon homeostasis, since 5-CH3-H4PteGlu is committed toward methionine biosynthesis, and unnecessary accumulation of this cofactor results in the depletion of the Cl folate pool and increased inhibition of SHMT (Matthews et al., 1982). 5- CHO-H,PteGlu, is formed by the hydrolysis of 5,10-CH+- H,PteGlu, by SHMT (Stover and Schirch, 1990). No known formyl transfer reactions utilize this form of the coenzyme. The role of this coenzyme is not known, but the monoglutamate derivative of the coenzyme has been demonstrated to be an inhibitor of rabbit liver cSHMT (Schirch and Ropp, 1966).

Frieden et al. (1980) have described three classes of competitive inhibitors: those which inhibit instantaneously, those which inhibit rapidly, followed by a slow conformational change, and those which inhibit slowly. Classical competitive inhibitors show a high affinity for the active site of the ground state enzyme, whereas slow binding inhibitors show a high affinity for the intermediate state enzyme. Slow binding inhibition is characterized by an initial weak binding to the ground state enzyme, followed by a slow conformational change. In general, this type of inhibition is considered more physiologically relevant since upstream accumulation of the substrate cannot relieve the inhibition brought about by the conformational change (Morrison and Walsh, 1987).

Both the high affinity of 5-CH3-H4PteGlu and 5-CHO- H,PteGlu for SHMT and their high concentrations in the cell suggest that these cofactors are capable of regulating the activity of this enzyme in vivo. In this paper, we investigate the interaction of the monoglutamate and triglutamate forms of the cofactors (5-CH3-H4PteGlu1+3, 5-CHO-H4PteGlul + 3 )

with SHMT. These studies show that there are significant differences in the mechanism of binding of the triglutamate forms of these two N’-reduced folates to SHMT.

EXPERIMENTAL PROCEDURES

Materials-Glycine, aminomethylphosphonate, DL-allothreonine, MgATP, NADPH, folic acid, glucose 6-phosphate, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, and 2-mercaptoethanol were purchased from Sigma. (6R,S)-H4PteGlul and (6R,S)-S-CHO- H,PteGlu, were purchased from Fluka and used without further purification. Pteroylpolyglutamates were purchased from Dr. B. Schircks laboratory in Switzerland and reduced to the tetrahydro

1543

1544 Serine Hydroxymethyltransferase

form as described by Strong et al. (1987). The reduced pteroylpolyglutamates were purified on DEAE-Sephadex according to the method described by Strong and Schirch (1989). (6S)-5-CHO- H,PteGlu,l were prepared as described previously (Stover and Schirch, 1990). (6S)-5-CH:l-H,PteGlu3 was prepared by incubating a 1.0 mM solution of (6S)-H,PteGlu3 with a 1.5 M excess of formaldehyde for 6 min in 10 mM KMES, pH 7.0., followed by the addition of a 20-fold molar excess of sodium borohydride. The solution was concentrated and purified as described previously (Strong and Schirch, 1990). All other chemicals were purchased from Fisher.

Rabbit liver Cl-THF synthase, CH‘THF synthetase, and the mi- t,ochondrial and cytosolic isozymes of SHMT, and Escherichia coli SHMT were purified to homogeneity as described previously (Schirch and Peterson, 1980; Villar et al., 1985; Hopkins and Schirch, 1984; Schirch et al., 1985).

Determination of Enzyme and Folate Derivative Concentrations- The concentration of all enzymes was determined by their absorbance at. 280 nm (Gavilanes et al., 1982; Schirch et al., 1985; Villar et al., 1985; Hopkins and Schirch, 1984). The concentrations of stock H,PteGlu, solutions were determined by a coupled enzymatic assay using cSHMT and 5,10-CH2-THF dehydrogenase. The increase in absorbance at 340 nm, resulting from the reduction of NADP’ and the formation of 5,10-CH+H4PteGlu, has an extinction coefficient of 7200 M-’ cm” at pH 7.3 (Schirch, 1978). The concentration of 5- CHO-H,PteGlu, stock solutions was determined by incubating the cofactor with 5,10-CH+-THF synthetase in 1 mM MgATP, 50 mM KMES, pH 6.0, and measuring the increase in absorbance a t 360 nm. The extinction coefficient of the product CH+-H,PteGlu a t 360 nm is 25,100 M” cm” (Blakley and Benkovic, 1984). 5-CHs-H,PteGlu, concentrations were determined using the extinction coefficient of :32,000 M” cm” at 290 nm, pH 7.0 (Blakely and Benkovic, 1986). All concentrations of folate coenzymes are recorded as the concentration of the physiological stereoisomer.

Determination of Dissociation Constants for N5-H,PteClu,-Dis- sociation constants for 5-CHO-H4PteGlu, and 5-CHR-H,PteGlu, were calculated from Scatchard plots by titrating a cSHMT-glycine solu- f.ion with NS-H4PteGlu, (Scatchard, 1949). A solution of 50 mM KMES, 0.4 pM SHMT, and 50 mM glycine was titrated with increasing concentrations of the coenzyme in a 10-cm pathlength cell. The increase in absorbance a t 502 nm, representing the formation of the SHMT. Gly .5-CHO-HsPteGlu, ternary quinonoid complex was used a s a measure of the bound cofactor (Strong and Schirch, 1989). Using an extinction coefficient of 40,000 M” cm” for the 5-CHO-HIPteGlu3 ternary complex, and 50,000 M” cm-’ for the 5-CH3-H4PteGlu3 ternary complex, the concentrations of free and bound N”H,PteGlu

previously described (Strong and Schirch, 1989). were determined from the expression for the dissociation constant as

Inhibition of cSHMT with N5-H4PteClu,-Inhibition constants of allothreonine cleavage for the inhibitors 5-CH:+-H4PteGlu, and 5- CHO-H,PteGlu, were determined from initial velocity measurements using a coupled assay syst,em with alcohol dehydrogenase as described previously (Schirch et al., 1977). Assays were performed by determining the rate of decrease in absorbance at 340 nm after addition of 170 pmol of cSHMT to a 1-ml cuvette containing 150 mM L-allothreonine, 0.15 mM NADPH, 0.05 mg of alcohol dehydrogenase, and between 0 and 200 p~ N’-H,PteGlu,, in 50 mM KMES, pH 7.0. The concentration of the inhibitor was increased until the rate of decrease in absorbance a t 340 nm became independent of inhibitor concentration.

Inhibition constants for the SHMT catalyzed aldol cleavage of serine by the inhibitors 5-CHs-HIPteGlu:+ and 5-CHO-H4PteGlun were determined by the method of Dixon (Segal, 1975) using the CI- THF synthase-coupled assay (Schirch and Quashnock, 1981). Reac- tions were initiated by the additicn of 25 pmol of cSHMT to a solution containing 40 p M L-serine, 0.5 mM NADP’, H,PteGlu, (5- 40 p ~ ) , 0.5 p~ CI-THF synthase, and 5 mM 2-mercaptoethanol in 50 mM KMES, pH 7.0, and the rate of increase in absorbance a t 340 nm determined.

Determination of initial rates of serine aldol cleavage by the c S H M T . G I ~ . ~ - C H O - H , P ~ ~ G I U : ~ ternary complex were performed by

p~ cSHMT, 500 p~ glycine) and the cSHMT.Gly. 5-CHO-H4PteGlu:, preparing stock solutions of the cSHMT.Gly binary complex (100

ternary complex (100 pM cSHMT, 500 p~ glycine, 150 pM W,PteGlu:J and diluting the enzyme complexes 4000-fold into a serine assay solution. Absorbance measurements at 502 nm showed that the stock solution was over 98% in the ternary complex. After dilution in the assay solution the rate of increase in absorbance a t 340 nm was determined. The serine assay contained 10 mM L-serine, 0.5 pM CI- THF synthase, 0.5 mM NADP’, 40 p M H,PteGlu,, 5 mM 2-mercap-

toethanol in 50 mM KMES, pH 7.0. Recovery of activity experiments were performed as described above with the exception that the ternary complex reaction was diluted 4000-fold into a serine assay, minus the substrate H4PteGlul. After incubation of the diluted ternary complex in the serine assay solution for various time periods, the reaction was started by the addition of H,PteGlu, and the rate determined as described above.

Rapid Reaction Kinetics-First order rate constants describing the rate of formation and breakdown of the SHMT. Gly.NS-H,PteGlu, + : I

quinonoid complex at 502 nm were obtained using a stopped-flow spectrophotometer from Kinetic Instruments Inc. Rate constants for quinonoid formation, as shown in Table 11, were obtained from measurements of the increase in absorbance a t 502 nm with time after flowing a solution containing 10 FM SHMT, 50 mM glycine in 50 mM KMES against a solution of 200 p~ (6-R,S)-N5-H4PteGlu,, 50 mM glycine in 50 mM KMES. Alternatively, cSHMT-N5- H4PteGlu, solutions were flowed against N5-H4PteGlu,-Gly solutions. Absorbance uersus time data were curve-fitted by single or double exponential algorithms as described previously (Shostak and Schirch, 1988). For the determination of rate of rotamer interconversion, a solution of 50 p~ SHMT and 50 mM glycine in 50 mM KMES, pH 7.0, was flowed against a solution of 4 p~ (6R,S)-5-CHO- H4PteGlul, and 50 mM glycine in the KMES buffer. All reactions performed in the stopped-flow instrument were done at 30 “C.

The rate of dissociation of glycine from the ternary complex was determined by flowing a solution of 50 mM KMES, pH 7.0, 5 mM glycine, 50 p~ N”-H,PteGlu,, and 10 p~ SHMT against a solution containing 50 mM KMES, pH 7.0, 100 mM aminomethylphosphonate and 50 p~ N5-H4PteGlu,. The decrease in absorbance a t 502 nm was recorded as a function of time. N‘--H,PteGlu, dissociation rates were obtained in a similar manner using a 20-fold molar excess of folic acid as the competitive binding ligand.

RESULTS

Spectra of the cSHMT. Gly ‘ N”-H,PteGlu,, Ternary Com- plexes-SHMT contains pyridoxal-P bound as an internal aldimine at the active site (Scheme 1, st ructure I ) . The absorbance properties of this coenzyme have enabled it to be used as a reporter group in the determination of the mechanism of this enzyme (Schirch, 1982). Scheme 1 shows the current accepted mechanism for the observable intermediates. SHMT catalyzes an exchange reaction of groups located on the 2s-position of serine and glycine, which are shown as R and R‘ in Scheme 1. When the enzyme is saturated with glycine, three complexes can be observed by their absorption maxima a t 343, 425, and 495 nm. These correspond to structures ZIZ, IV, and V in Scheme 1, respectively. The addition of H,PteGlu to the enzyme-glycine binary complex has been shown to shift the equilibrium toward structure V, which is referred to as the quinonoid complex (Schirch and Ropp, 1967). The ternary SHMT. Gly- . H4PteGlu, complex has an apparent extinction coefficient of 40,000 M” cm” (Strong and Schirch, 1989). However, because of the impurities present in H4PteGlu solutions, and their intense absorption below 400 nm, the effect of H4PteGlu on the distribution of the complexes between structures IZZ, ZV, and V is unknown.

Fig. 1A (curoe 2 ) shows the absorption spectra of the SHMT.Gly complex at pH 7.0. Curve 3 shows the effect of adding a 1.5 M excess of 5-CHO-H,Pt,eGlua, with respect to the enzyme, which results in the formation of the ternary SHMT. Gly . 5-CHO-H,PteGlu3 complex in greater than a 98% yield. Because of the purity, stability, and tight binding of the triglutamate, you can determine from the spectrum if s t ructures IIZ and IV are still present in the ternary complex or whether the ternary complex is essentially all structure v (Scheme 1). The retention of considerable absorbance at both 343 and 425 nm suggests t.hat structures III and IVare present in significant concentrations in the ternary complex. This is further comfirmed by the data in Fig. 18 which shows the CD spectra of the unliganded cSHMT (curve I ) , the cSHMT. Gly binary complex (curve 21, and the cSHMT.Gly.5-CHO-

Serine Hydroxymethyltransferase 1545

SCHEME 1

343nm 425 nm 500 nm

m Ip Y 423 run

I II

0 2 5 , I

0 .os

nrrlaom N

FIG. 1. Absorbance and CD spectra of cSHMT and its complexes with glycine and (6S)-5-CHO-H4PteGlu3. A, absorbance spectra of cSHMT saturated with glycine (curue 2 ) and cSHMT saturated with glycine and (6S)-5-CHO-HdPteGlu.1 (curue 3 ) . 8, CD spectra of cSHMT (curue I ) , cSHMT saturated with glycine (curue 21, and cSHMT saturated with glycine and (6S)-5-CHO-H,PteGlu3.

H ,p teGl~ :~ ternary complex (curue 3 ) . CD spectra show less interference from 5-CHO-H4PteGlu,, because it selects only for chromophores bound to the enzyme that exhibit optical activity. The addition of saturating amounts of 5-CHO- H4PteGlu3 to the cSHMT . Gly complex results in the reduction of structures III and IV by about 40-50%. However, it is clear that significant concentrations of complexes 111 and IV are still present in the SHMT.Gly.5-CHO-H4PteGluS complex.

These studies were repeated with 5-CH:3-H4PteGlu3, and both the absorption and CD spectra were similar to those recorded in Fig. 1. However, as reported previously by Mat- thews et al. (1982) for pig liver SHMT, the apparent molar absorptivity for the cSHMT. Gly.5-CH:l-H4PteGlua quinonoid species is 50,000 M" cm". This shows that in the ternary complex the equilibrium distribution lies more in favor of structure V (Scheme 1) with 5-CH3-H4PteGlu3 than with 5- CHO-H,PteGlu,.

Dissociation Constants of N"-H4PteG1u3-Previously, the intense absorption of the quinonoid structure of the SHMT ternary complexes has been used to determine the Kd values of glycine and unsubstituted and N5-substituted H,PteGlu, (Schirch and Ropp, 1966; Matthews et al., 1982). These studies have shown that substitution at the N 5 position with either a methyl or formyl group has little effect on the affinity of the reduced folate for the SHMT.Gly complex ( K d = 10- 15 p ~ ) . There was, however, a 5-fold synergism detected for the binding of glycine and the reduced folate compounds (Schirch and Ropp, 1966). The addition of glutamate residues

TABLE I Thermodynamic parameters associated with the stability of the

enzyme. Gly . N'-H,PteClu, ternary complex

Ternary complex K d NS-Folate

Glycine N'-Folate"

P M P M

cSHMT.Gly.5-CHO-H,PteGlul 1600 10.0 * 1.0 cSHMT.Gly.5-CHO-H4PteGIu:, 32 ? 5 0.2 5 0.1 0.2 5 0.1 cSHMT.Gly.5-CH:,-H,PteGlul 520' 11' cSHMT . Gly . B-CH,,-H,PteGlu:, 34 i 5 0.4 & 0.1 0.5 & 0.1 * Determined from the absorbance of the ternary complex of 502

'Determined from initial velocity studies using H,PteGlu, and nm.

serine as substrates. Values from Schirch and Ropp (1966).

to unsubstituted H,PteGlu,, does not significantly alter the affinity of the rabbit liver cSHMT- H,PteGlu binary complex for glycine (Strong and Schirch, 1989). However, as shown with pig liver SHMT, the 5-CHs-H4PteGlu6 binary complex displays a 200-fold increase in affinity for glycine compared with the affinity with the monoglutamate SHMT-complex (Matthews et al., 1982). The effect of increasing glutamate chain length on the affinity of the SHMT. 5-CHO-H4PteGlu, complex for glycine has not been reported previously.

The effects of polyglutamate chain length on the affinity of both N " - H , P ~ ~ G ~ u , + : ~ derivatives and glycine for rabbit cSHMT were determined by observing the increase in absorbance a t 502 nm as a function of increasing ligand concentration (Table I). The dissociation constants for the trigiutamate derivatives of 5-CHO-H,PteGlus and 5-CH3-H4PteGlu3 are similar, with their dissociation constants being decreased about 30-fold compared with the monoglutamate derivatives. The dissociation constants for glycine are reduced 50- and 15- fold, respectively, for SHMT. 5-CHO-H4PteGlu3 and 5-CH:]- H,PteGlu:,. The similarities in the binding constants and effect of the polyglutamate chain for 5-CHO-H4PteGlu:, and 5-CH3-H,PteGlu,, suggests that these compounds interact with the cSHMT . Gly complex by the same mechanism.

The effectiveness of 5-CHO-H4PteGlu:{ and 5-CHs- H4PteGlu:? in inhibiting the cleavage of serine with H,PteGlu, as the variable substrate was also determined. Both compounds were found to be effective competitive inhibitors of H,PteGlu, in the reaction. The Ki values for the two triglutamate forms were found to be essentially identical to their K d values (Table I). The initial velocity patterns showed that the inhibition was immediate for both the 5-CHs-H4PteGlul + R

and 5-CHO-H4PteGlu, derivatives. However, the 5-CHO- H4PteGluB appeared to display a slow time dependent increase in inhibition, a property which was not displayed by the 5- CH3-H4PteGlu3 derivative. This slow increase in inhibition could not be quantitated for two reasons. First, to show competitive inhibition, less than saturating levels of HIPteGlu, were used as substrate in the assay. The concentration of the coenzyme decreased during the assay resulting


in curves which stayed linear for only about 20 s. Second, H4PteGlu undergoes oxidation nonenzymatically in the assay solution giving rise to oxidation products which absorb a t 340 nm. This results in a nonlinear base line which is difficult to accurately subtract from the reaction curve over a period of 1 to 2 min.

In order to better quantitate this apparent slow time- dependent inhibition, a series of enzyme activity recovery experiments were performed (Fig. 2). A concentrated solution of cSHMT (100 p ~ ) , glycine (500 PM), and 5-CHO-H,PteGlu3 (150 p M ) was prepared and calculated to be greater than 98% as the ternary complex as determined from its absorption at 502 nm. This solution was diluted 4000-fold into a reaction mixture containing saturating concentrations of serine and H4PteGlul. The addition and mixing took 4 s. The increase in absorbance was linear, but exhibited only 52% of the activity of a control in which 5-CHO-H4PteGlu3 had been omitted from the concentrated SHMT. Gly solution (Fig. 2 A ) . In a second experiment, the concentrated solution of the SHMT. Gly . 5-CHO-H4PteGlu3 complex was diluted in an assay solution which did not contain H,PteGlu as a substrate. The folate substrate was added to initiate the catalytic reaction after several seconds. The results of this experiment are shown in Fig. 1B. If the folate substrate was added 8 s after the dilution of the ternary complex the rate was 66% of the control value. If the folate substrate was added either 16 s or 3 min after the dilution the activity was 82 and 100% of the control, respectively. When these experiments were repeated with either 5-CHO-PteGlul or 5-CH3-H4PteG1u3, no inhibition of activity was observed even in the absence of any incubation time after the dilution of the ternary complexes.

The results in the previous paragraph suggest that both 5- CHO-H,PteGlu, and 5-CHs-H,PteGlul+3 are classical competitive inhibitors of cSHMT as shown by reaction (1). How-

-0 5 10 15 seconds

FIG. 2. Rate of recovery of activity when the cSHMT*Gly*5-CHO-H4PteGlu3 ternary complex is diluted 4000-fold into an assay solution. A, the initial rate of serine cleavage by 25 pmol of cSHMT (-) and 25 pmol of the cSHMT. Gly . 5-CHO-H,PteGlu3 ternary complex (- - - -). B, the time-dependent recovery of activity after incubation a t different time periods solutions without H,PteGlu. The numbers above each trace represent time of incubation in seconds after dilution, but before starting the reaction by addition of the substrate H4PteGlu.

ever, the results suggest that 5-CHO-H4PteGlu3 inhibition may fit a two step model with an initial rapid onset of inhibition, followed by a slower time-dependent increase in inhibition as shown by reaction (2). The data shown in Fig. 2 suggest that the equilibrium between E . I and E . I* is about 1. This would explain why only 52% of the activity of the ternary complex is recovered rapidly after dilution and the remaining 48% activity is recovered only after a several min- ute incubation.

E + I- E.1

fast slow

fast (1)

(2)

The competitive inhibition of serine cleavage by both 5- CHO- and 5-CH3-H4PteGlu3 shows that the inhibitors can bind to the free enzyme. Previously, inhibition studies of allothreonine cleavage by 5-CH3-H4PteGlul showed that 5- CH,-H,PteGlu, could also bind to the enzyme-Gly complex (structure V, Scheme 1) (Schirch et al., 1977). These inhibition studies of allothreonine cleavage, which do not require a folate substrate, were repeated with N5-H4PteGluI and N5- H4PteGlu3. As shown in Fig. 3, increasing concentrations of 5-CHO-H4PteGlul and 5-CH3-H4PteGlu3 result in inhibition with a limiting value for kcat of 0.4 s-I. This value was also found previously for the limiting rate of allothreonine cleavage in the presence of 5-CH3H4PteGlu, (Schirch et a!., 1977). In these previous studies the limiting rate at a saturating concentration of 5-CH3-H4PteGlul was shown to be the rate at which glycine dissociates from the ternary complex. Matthews et al. (1982) found similar results using pig liver SHMT and went on to show that the limiting rate of allothreonine cleavage in the presence of high concentrations of all polyglutamate forms of 5-CH3-H4PteGlu, was independent of the number of glutamate residues. The conclusion drawn from these results was that the increased affinity of the polyglutamate forms must be a function of the k,, rate for 5-CH3-H4PteGlu, (Matthews et al., 1982).

When 5-CHO-H4PteGlu3 was used as an inhibitor of allothreonine, a different result was observed as shown in Fig. 3. The final kc,, at saturating inhibitor concentration was 0.05 s" as opposed to the value of 0.4 s" observed with 5-CHO- H4PteGlul. This shows that the number of glutamate residues on this form of folate does affect the dissociation rate of glycine from the ternary complex.

Rapid Reaction Kinetics Associated with Quinonoid Forma- tion and Breakdown-A more direct approach to defining the factors affecting the binding of inhibitors to an enzyme is to examine the kinetic constants associated with formation and breakdown of the E .I complex using rapid reaction tech-

E + I - E . 1 L__ E.I*

P-H4Pte61u Inmoles)

FIG. 3. Inhibition of the cSHMT catalyzed aldol cleavage of allothreonine by increasing concentrations of N6-substituted derivatives of H4PteGlu,. m"., 5-CHO-H4PteGlul; A-A, 5-CH3- H4PteGlua; e"., 5-CHO-H4PteGlur.

Serine Hydroxymethyltransferase 1547

niques. Table I1 summarizes the rate of formation and breakdown of the quinonoid complex with the mono and tri glutamate forms of 5-CHO-H4PteGIu and 5-CH3-H4PteGlu. All experiments measuring the apparent rate of quinonoid formation were performed with at least a 10-fold excess N s - H,PteGlu, over SHMT. With each form of the enzyme stud- ied, the apparent rate of quinonoid formation and breakdown was about an order of magnitude slower with 5-CHO- H,PteGlu, +:, as compared with 5-CH3-H4PteGlu,+:3. The quinonoid breakdown rates were essentially the same when either glycine or N"-H4PteGlu, competing ligands were flowed against the ternary complexes, with the exception of H228D SHMT (Table 11). For H228D SHMT, the apparent k , for the NS-H,PteGlul is 4-fold faster than the glycine apparent k, (Table 11). This observation was not pursued further in this study. These results suggest that the limiting k, values for glycine and Ns-H,PteGlu, are the result of interconversion of enzyme complexes rather than dissociation of the ligand from the enzyme.

There were two notable features associated with quinonoid formation upon the addition of the two additional glutamate residues of 5-CH,+-H4PteGlun and 5-CHO-H4PteGlu3. First, the absorbances at 502 nm versus time traces for both cofactors were biphasic, with both phases being of approximately equal amplitude (Table 11). The biphasic character remained even in the presence of excess cofactor with respect to enzyme. Second, although the rate of quinonoid formation and breakdown was decreased by less than 2-fold when 5-CH3- H4PteGlu1 was replaced with the triglutamate, the rate of quinonoid formation and breakdown with 5-CHO-H4PteGlu3 was almost an order of magnitude slower compared with the monoglutamate derivative. The rates of formation and dissociation of the SHMT. Gly .N5-H4PteGlul quinonoid complex were also determined for rabbit liver mitochondrial SHMT, E. coli SHMT, and a mutant form of the E. coli enzyme H228D.

Euidence That Only One Rotamer of 5-CHO-H4PteGlul Binds to the SHMT-Gly Complex-The solution structure of 5-CHO-H,PteGlul has been determined by both "C and 'H NMR spectroscopy and found to be an equilibrium mixture of two slowly interconverting forms (Feeney et al., 1980, Poe and Benkovic, 1980). These two forms were attributed to the partial double bond character of the C"-N5 formamide bond, creating two rotamers present a t a ratio of 2.35 to 1.0 at 25 "C. The N 5 formyl group is bent out of the plane of the pteridine ring and has been demonstrated to lie on the same side of the ring as the C6 hydrogen (Poe and Benkovic, 1980). The more abundant rotamer has the formyl proton oriented in the same

TABLE I1 Rapid reaction kinetic constants associated with the formation and

breakdown of the quinonoid ternary complex

ternary complex ternary complex

complex 5-CHO- 5-CH:,- 5-CHO- 5-CH,-

Formation of Breakdown of

Enzyme. Gly "l;,"' residues H,PteGlu, H4PteGlu, H,PteGlu, H,PteClu,

( k 0 ( k , ) ( k , ) " (k,)" S" S"

cSHMT.Gly 1 0.50 cSHMT.Gly 3 0.05, 0.19'' 2.0, 6.70' 0.04 0.55

4.80 0.39 0.90

mSHMT-Gly 1 0.15 3.50 0.10 0.63 E. coli SHMT 1 0.05 3.10 0.04 0.59

. Cly H228D.Gly 1 2.20 12.70 0.36 3.90

"Determined from the breakdown of the SHMT.Gly.N5- H,PteClu, quinonoid complex with folic acid as the competing ligand.

'The rate of formation of the quinonoid complex was biphasic. The amplitudes of the two phases were similar.

plane as the keto group of C4, which is deshielded in the 'H NMR spectra. These spectra also display deshielding of the C" proton due to its proximity to the formyl carbonyl group. The tetrahydropyrazine ring exists in a half chair conformation with the CG proton in the equatorial position (Poe and Benkovic, 1980).

The relatively slow rate of formation and high apparent extinction coefficient of the quinonoid species permits sensi- tive and accurate determinations of the rate of formation and breakdown of this complex using stopped-flow spectroscopy. During these studies, it was observed that quinonoid formation was biphasic when 5-CHO-H4PteGlul was flowed against an excess of the SHMT. Gly binary complex. The slow phase of quinonoid formation with 5-CHO-H4PteGlul had a half- life of approximately 2 s-' at 40 "C, which is similar to the rate of rotamer interconversion demonstrated by Feeney et al. (1980).

Performing the identical experiment, described in the previous paragraph, but with a 10-fold excess of 5-CHO- H4PteGlul over the SHMT. Gly binary complex results in a single exponential reaction with a first order rate constant equal to that of the previously described rapid phase. This suggests that the slow phase is not a property of the enzyme. If the rate of the slow phase represents rotamer interconversion, then this rate should be independent of the source of SHMT. Unfortunately, mSHMT and E. coli SHMT could not be used in this study, since the rate of quinonoid formation is less than the rate of putative rotamer interconversion (Table 11). We were, however, able to use a mutant form of the E. coli enzyme, H228D SHMT, which does display a rapid formation of the quinonoid complex with excess 5-CHO- H4PteGlul (Table 11). When quinonoid complex formation was determined with excess SHMT. Gly complex, a biphasic reaction was observed with a slow phase having the same rate as observed with cSHMT.

Fig. 4 shows the rate of SHMT-Gly-5-CHO-H4PteGlul quinonoid complex formation at 50'2 nm with a 10-fold excess of E. coli H288D SHMT-Gly binary complex over 5-CHO- H4PteGlu, using a stopped-flow apparatus. The slower rate in this biphasic curve is identical for both cytosolic and H228D E. coli SHMT over the temperature range 25-40 "C, suggesting that it represents the first order rate of rotamer interconversion. Under the same conditions of excess enzyme, neither 5-CH3-H,PteGlul nor H4PteGlul exhibit biphasic kinetics upon forming the quinonoid complex with either the cytosolic of H228D SHMT. These results suggest that SHMT is capable of forming the quinonoid complex with only a single

701 I

f :[ 20

10

g ::;I \ I -4.0 -5.0

0.31 0.32 0.33 0.34 0.35 0.36 111 x 100

of I 0 5 10 15 20

seconds

FIG. 4. Rate of formation of E. coli H228D SHMT*Gly*5- CHO-H4PteGlul quininoid complex with limiting 5-CHO- H4PteGlul by stopped-flow spectrophotometry. A solution of 50 p M H228D SHMT in 50 rnM glycine was flowed against 4 1 M 5-CHO- H4PteGlul and monitored a t 502 nm. The solid line is a curve fit for a two exponential reaction. The inset shows the change in the slower rate as a function of temperature, plotted by the method of Arrhenius.


rotamer of 5-CHO-H,PteGlu,. The relative amplitude of the fast and slow phases for both cSHMT and H228D SHMT are 2.5 to 1 at 30 "C, consistent with SHMT binding the more populated rotamer where the C" and C4 carbonyl oxygens are maximally separated.

Previous 'H NMR studies of 5-CHO-H4PteGlul demonstrated that the relative distribution of the two rotamer populations was temperature dependent and the associated equilibrium thermodynamic parameters were determined (Feeney et al., 1980). The changes we observed in relative amplitude of the fast and slow phases with both enzymes as a function of temperature (2.5:l a t 30 "C and 3.3:l at 40 "C) is consistent with the change in the relative populations of the two rotamers (2.35:l at 25 "C and 4.66:l at 77 "C) determined by NMR (Feeney et al., 1980).

The rate of rotamer interconversion as a function of temperature was determined and plotted by the method of Ar- rhenius (Fig. 4, inset). The data show the slow phase of quinonoid ternary complex formation, associated with rotamer interconversion, over the temperature range 10-40 "C for the H228D enzyme. These values were identical for the cytosolic enzyme over the temperature range 25-40 "C. Below 25 "C, quinonoid formation became rate limiting for cSHMT. From this plot, the E, for rotamer conversion from the less populated to the more populated state was determined to be 24 kcal/mol.

DISCUSSION

The conversion of 5,lO-CH'-H4PteGlun to 5-CHO- H,PteGlu, by SHMT is an unusual example of enzymatic regulation whereby an enzyme, displaying a second unrelated enzymatic activity, produces an effective inhibitor of its principal physiological reaction (Stover and Schirch, 1990). The high concentrations of 5-CHO-H4PteGlu, found in certain cells, particularly in seedlings, suggests that this compound may play a major role in regulating SHMT activity. In order to better define the role of this compound in viuo, we have further investigated the properties of the SHMT. Gly.5- CHO-H4PteGlu3 ternary complex and the related SHMT- Gly . 5-CH3H4PteGlu3 ternary complex.

The results reported in this study show that the triglutamate forms of 5-CHO- and 5-CH3-H4PteGlu3 exhibit similar Kd and Ki values for cSHMT. The similarity of the constants suggests that these two inhibitors are of the classical competitive type and inhibit SHMT by rapidly forming an E.1 complex as shown in Reaction 1. However, closer inspection of the kinetics of inhibitor binding and dissociation clearly show that the polyglutamate forms of 5-CHO-H4PteGlun inhibit by a mechanism that is different than the inhibition by 5-CHO- and 5-CH3-H4PteGlul and 5-CH3-H4PteGlu3. Reac-

tion 1 cannot explain the results on the rate of formation and breakdown of the quinonoid complex and the inhibition of serine catalysis by 5-CHO-H4PteGlu2.

We conclude that forming the SHMT. Gly .5-CHO- H4PteGlu3 ternary complex occurs by a different mechanism than forming the ternary complex with 5-CH3-PteGlu3. We also conclude that the polyglutamate chain of 5-CHO- H4PteGlus plays some role in the binding process, which does not occur with the 5-CH3-H4PteGlu3 cofactor. One model, which explains most of the results for forming the SHMT. Gly.5-CHO-H4PteGlu3, is shown in Scheme 2. In this mechanism, 5-CHO-H4PteGlu:3 binds relatively rapid to the cSHMT. Gly complex forming the geminal diamine and external aldimine complexes (structures IIZ and IV in Scheme 1). This is followed by a much slower conversion to the quinonoid complex absorbing at 502 nm (structure V in Scheme 1). Furthermore, considering the slower rate of formation, the ratio of ks/& appears to be near 1. This would mean that in the ternary complex, about half of the complex resides in the quinonoid form which is in slow equilibrium with the other half of the complex. This model is consistent with several observations. First, the spectral properties of the ternary complex recorded in Fig. 1 suggest that significant concentrations of the geminal diamine and external aldimine complexes are present. Although we cannot determine quan- titatively the exact relative concentrations of structures ZIZ, IV, and V, it is possible to estimate that only about 50% of the ternary complex is present as structure V. Second, the model in Scheme 2 explains why when the ternary complex is diluted in an assay solution, only 52% of the activity is immediately recovered and the remaining 48% is recovered in a time frame which is consistent with the value of k6 (Fig. 2 and Table 11). Third, the rate of formation of the quinonoid complex as determined by the increase in absorbance a t 502 nm is slow (and biphasic) with a half-life for the slow-phase of 14/s ( k s + k6). However, the rate of inhibition of serine cleavage by 5-CHO-H4PteGlu3 appears to show no lag phase. This is explained by the fact that most of the inhibition occurs by the rapid formation of the ternary complex in the geminal diamine and external aldimine complexes and the rapid phase of forming the quinonoid complex ( bh = 3 s) (Table 11). Since the initiation of the inhibition assay takes about 4 s, this means that about 70% of the ternary complex has already been formed when absorbance versus time data is recorded. The serine assay does not stay linear long enough to clearly determine that the remaining 30% inhibition occurs with a tw of 14 s. Fourth, the model in Scheme 2 predicts that the value of Ki* should be similar to the value of Ki. This is because the values of ks and k6 are nearly equal. We cannot accurately determine the value of kg because the rate of formation of the

FAST

k ? cSHh4T. Gly + 5-CHO-H P t e G l u - cSHMT. Gly 5-CHO-H4PteG1un 4 "7

SCHEME 2

k 6 11 SLOW

cSHMT. Gly 5-CHO-H4PteG1un e

(Quinonoid)

Serine Hydroxymethyltransferase

CSHMT- s e t . n P t c G I u n 4

1549

SCHEME 3

k = a.4sec- c a t 5.10. C ~ - ~ P I C G I U ,

" 1 k = 0.19.0.05 sec k =63, 2.0 w - f f

ESHMT Gly' S - C H O - H P t c G l u cSM* GlY,- ESHMT. G l y 5-CIfj-HqPtcGlu 8

4 3 - 3

quinonoid complex is biphasic. The value of K,* should be similar to the value of Kd determined by titrating the SHMT. Gly complex with 5-CHO-H4PteGlu3. The value of Ki is the inhibition constant determined from the initial velocity studies in the cleavage of serine. The values for both Ki and Ki* were determined to be 0.2 ~ L M (Table I). However, they both have errors of f O . l pM.

The model does not explain why the rate of formation of the quinonoid complex is biphasic with 5-CHO-H3PteGlu3. Nor is it clear why in the recovery of activity when the ternary complex is diluted into an assay mixture that the rate remains linear with time for at least 15 s (Fig. 3). One would predict that the rate should increase due to the conversion of the quinonoid complex to free enzyme (t1,* = 17 s). One factor which may contribute to an explanation of why the rate does not increase is that in the assay one of the intermediates is the SHMT.Gly complex which has a high affinity for the diluted 5-CHO-PteGlua (Fig. 2).

Typical slow binding inhibitors are substrate intermediate analogs which have a higher affinity for a catalytic intermediate state of the enzyme than the ground state enzyme. H4PteGlul + 3, 5-CH3-H4PteGlul + 3, and 5-CHO-H4PteGlul + 3

all exhibit rapid binding to the SHMT. Gly complex. This is a property consistent with competitive ground state inhibitors. However, the three coenzymes differ in their rates in forming quinonoid complexes (Scheme 3). Compared to kc,, in the conversion of serine to glycine (8.4 s-'), formation of the H4PteGlu, quinonoid complex is rapid (875 s-') (Schirch, 1975), whereas formation of the 5-CH3-H4PteGlu, quinonoid complex is the same order of magnitude (4.8 s-'). Only formation of the 5-CHO-H4PteGlus quinonoid complex exhibits slow formation (0.05 s-'). The slow apparent rate of inhibitor binding defines 5-CHO-H4PteGlu3 as an intermediate state inhibitor (Frieden et al., 1980). Based on these criteria, the 5- CHO-H,PteGlu, quinonoid complex best mimics the catalytic

A B

SCHEME 4

k, -0.04see-1 =OS5 sec - 1

k f = 8 7 5 u c - 1

c S H M T m G l y Y P t ~ O l u l Q

intermediate, suggesting that the cSHMT. Gly .5-CHO- H4PteGlus quinonoid complex is (structure B, Scheme 4) a true intermediate-state analog of the catalytically competent cSHMT. Ser . H,PteGlu, ternary complex (structure A, Scheme 4). The slow apparent rate of binding of intermediate- state inhibitors is typically understood in terms of considerable conformational changes required to adjust the enzyme from the ground state to the intermediate state (Morrison and Walsh, 1987). On the other hand, the rate at which the folate dissociates from the quinonoid complex is reflective of the degree at which the intermediate state is stabilized. Only the 5-CHO-H4PteGlu3 quinonoid exhibited k, values consid- erably less than keat values.

In addition, there appears to be communication between the one-carbon and polyglutamate binding sites on the enzyme. This communication is suggested by the dramatic changes in the rate of quinonoid formation and breakdown between ternary complexes formed with 5-CHO-H,PteGlu, and 5-CHO-H4PteGlu3 (Table 11). By comparison, rate constants associated in quinonoid formation and breakdown with 5-CH3-H4PteGlu, and 5-CH3-H4PteGlu3 are very similar. The differences in the rates of quinonoid formation and breakdown for 5-CHO-H4PteGlul and 5-CHO-H4PteGlu3 complexes suggest that the formyl group binding at the one-carbon site on the enzyme confers changes in the conformation of the polyglutamate binding site. Therefore, the structural relationship between the enzyme one-carbon site and the polyglutamate binding site may be different between the ground-state enzyme and the intermediate-state enzyme.

Finally, as would be expected for an intermediate-state-like complex, the cSHMT. Gly . 5-CHO-H4PteGlul quinonoid complex shows specificity for a particular rotamer of 5-CHO- H4PteGlu,. The high energy of activation (24 kcal/mol) determined for rotamer interconversion is due to the partial double bond character of the N"-C" formyl bond, and this value is similar to that found for other compounds exhibiting rotamers (Lee and Querijero, 1985). If the 5-CHO-H4PteGlu, quinonoid species does closely mimic the SHMT. Ser. H4PteGlu, catalytic complex, then we can make predictions about the relative displacement of the serine hydroxyl group relative to the C4 carbonyl oxygen of H4PteGlu, (Scheme 4). A single rotamer state of 5-CHO-H4PteGlul has also been observed for the cofactor when bound to dihydrofolate reduc- tase (Birdsall et al., 1981). Like SHMT, this enzyme is specific for the rotamer with the C4 and C" carbonyl oxygens maximally separated.

One of the unexplained observations about the mechanism


of cSHMT is that N”-hydroxymethyl-H,PteGlu, is not a substrate for the enzyme (Schirch and Chen, 1973). The observation that cSHMT binds only one of the rotamers of 5-CHO-H,PteGlul may explain this observation. It seems probable that in solution, the N5-hydroxymethyl-H,PteGlu, may have the hydroxyl group pointing toward the C4 carbonyl where it can form a hydrogen bond. If this stabilizes this structure, then it would correspond to the rotamer of 5-CHO- H,PteGlu, which does not bind to the active site of cSHMT.

The product of serine catabolism, 5,10-CH2-H,PteGlu, is the principal entry point of one-carbon units into the one- carbon pool. In vivo, there are four enzymes capable of con- verting an amino acid substrate and tetrahydrofolate to 5,lO- CH2-H4PteGlu, all of which bind 5-CHO-H4PteGlu with high affinity. In the mitochondria, dimethylglycine dehydrogenase and sarcosine dehydrogenase have been demonstrated to bind both 5-CH,3-H4PteGlul and 5-CHO-H4PteGlu, with Kd values in the 1.5 PM range (Wittwer and Wagner, 1980). Bacterial sarcosine oxidase also displays high affinity for 5-CHO- H4PteGlu,, whereas no binding was detected for 5-CH:,- H,PteGlu, (Kvalnes-Krick and Jorns, 1987). The high concentration of these N”-substituted folates in the one-carbon pool suggests that these compounds are capable of regulating the entry of one-carbon units into the folate pool. Therefore, regulation of the supply of these folate derivatives may be critical in maintaining cellular homeostasis.

REFERENCES Birdsall, B., Burgen, A. S. V., Hyde, E. I., Roberts, G. C. K., and

Blakely, R. L., and Benkovic, S. J . (1984) in Chemistry and Biochem-

Chen, M. S., and Schirch, L. (1973) J. Biol. Chem. 248, 7979-7984 Feeney, J., Albrand, J. P., Boicelli, C. A., Charlton, P. A., and Young,

Feeney, J. (1981) Biochemistry 20, 7186-7195

istry of Folates, Vol. I, p. 80, John Wiley & Sons, New York

D. W. (1980) J. Chem. SOC. Perkin Trans. 2, 176-180

5311

257, 11431-11436

Frieden, C., Kurz, L., and Gilbert, H. (1980) Biochemistry 19, 5306-

Gavilanes, F., Peterson, D., and Schirch, L. (1982) J. Biol. Chem.

Hopkins, S.. and Schirch, V. (1984) J. Bid. Chem. 259, 5618-5622 Horne, D. W., Patterson, D., and Cook, R. J. (1989) Arch. Biochem.

Jencks, D. A,, and Matthews, R. G. (1987) J. Biol. Chem. 262,2485-

Kvalnes-Krick, K., and Jorns, M. S. (1987) Biochemistry 26, 7391-

Lee, H., and Querijero, G. (1985) J. Pharm. Sci. 74, 273-276 Matthews, R. G., Ross, J., Baugh, C. M., Cook, J . D., and Davis, L.

Morrison, J. F., and Walsh, C. T. (1987) Adu. Enzymol. Relat. Areas

Poe, M., and Benkovic, S. J. (1980) Biochemistry 19, 4576-4583 Scatchard, G. (1949) Ann. N. Y. Acad. Sei. 51,660-672 Schirch, L. (1975) J. Bid. Chem. 250, 1939-1945 Schirch, L. (1978) Arch. Biochem. Biophys. 189, 283-290 Schirch, L. (1982) Adv. Enzymol. Relat. Areas Mol. Biol. 53, 83-112 Schirch, L., and Ropp, M. (1967) Biochemistry 6, 253-257 Schirch, L., and Peterson, D. (1980) J . Bid. Chem. 255, 7801-7806 Schirch, L., and Quashnock, J. (1981) J. Bid. Chem. 256,6245-6249 Schirch, L., Tatum, C. M., and Benkovic, S. J . (1977) Biochemistry

Schirch, V., Hopkins, S., Villar, E., and Angelaccio, S. (1985) J.

Segal, I. H. (1975) in Enzyme Kinetics, pp. 109-111, John Wiley &

Shostak, K., and Schirch, V. (1988) Biochemistry 27, 8007-8014 Stover, P., and Schirch, V. (1990) J. Bid. Chem. 265, 14227-14233 Strong, W. B., and Schirch, V. (1989) Biochemistry 28, 9430-9439 Strong, W., Joshi, G., Lura, R., Muthukumaraswamy, N., and

Villar, E., Scbuster, B., Peterson, D., and Schirch, V. (1985) J. Bid.

Wittwer, A. J., and Wagner, C. (1981) J. Bid. Chem. 256,4102-4108

Biophys. 270, 729-733

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(1982) Biochemistry 21, 1230-1238

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Schirch, V. (1987) J. Bid. Chem. 262, 12519-12525

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