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Serine transhydroxymethylase: Mechanism of aldolase activation by folate
ARCHIVES
OF
BIOCHEMISTRY
AND
174,
BIOPHYSICS
Serine Transhydroxymethylase: Activation CLIFTON Department
of Biochemistry,
Mechanism by Folate
W. JONES,
Medical
University Received
(1976)
305-311
III, of
South
September
AND
of Aldolase
D. G. PRIEST
Carolina,
Charleston,
South
Carolina
29401
29, 1975
Affinity chromatographic methods have been developed to purify beef liver serine transhydroxymethylase. This enzyme catalyzes both the transfer of aldehydic groups from /3-hydroxy amino acids to tetrahydrofolate and the cleavage of p-hydroxy amino acids yielding the free aldehyde. Tetrahydrofolate, folate, and a quinazoline analog of isofolate were found to be a$.ivators of the p-phenylserine aldolase reaction catalyzed by serine transhydroxymethylase. Activation by folate was maxima1 at 20 FM, and higher concentrations diminished the activation. Evidence is presented suggesting that folate does not activate by providing an acceptor site for the aldehydic groups. Equilibrium binding studies showed that folate and tetrahydrofolate can bind the enzyme with essentially the same affinity. Double-reciprocal plots with P-phenylserine from steadystate kinetic experiments did not yield a l/u, intercept effect except at high folate concentrations. A mechanism is proposed in which folate binds readily to the enzymic active site, facilitating p-phenylserine binding. Folate is subsequently lost, at least partially, prior to product release and complete enzymic turnover.
Serine tran+ydroxymethyIase, L-serine:tetrahydrofolate 5, lo-hydroxymethyltransferase (EC 2.1.2.11, catalyzes the transfer of a hydroxymethyl group from serine to tetrahydrofolate (1) and also the cleavage of serine and other /3-hydroxy amino acids yielding glycine and the corresponding aldehydic product in an aldolasetype reaction (2). Several such P-hydroxy amino acids are substrates for the enzymes isolated from rabbit liver (3) and lamb liver (4). We have found folate, tetrahydrofolate, and a quinazoline analog of isofolate to be activators of the /3-phenylserine aldolase reaction catalyzed by bovine liver serine transhydroxymethylase. The aldolase reaction with threonine as substrate was not enhanced by folate under the conditions used. The relative contribution of folate to the structural suitability of the enzymic active site, as opposed to its participation as an acceptor for the aldehydic group, was investigated. Results of these studies are reported here and interpreted in terms of an enzymic mechanism of folate activation.
MATERIALS
’ Abbreviation 305
Copyright All rights
5: 1976 by Academic Press, Inc. of reproduction in any form reserved.
AND
METHODS
The EDTA, folic acid, nL-threo-P-phenylserine, Lserine, L-threonine, Coomassie brilliant blue R, pyridoxal5-phosphate, phenylhydrazine hydrochloride, and alcohol dehydrogenase were obtained from Sigma Chemical Company. Sepharose 4-B and CMSephadex’ were obtained from Pharmacia Fine Chemicals Company. Potassium phosphate, 1,6hexanediamine, sodium acetate, and dimethylformamide were obtained from Fisher Scientific Company. Ammonium sulfate was obtained from SchwarziMann. The I-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride was obtained from Pierce Chemical Company. The 2-amino-6-(4carbethoxybenzylamino)-4-hydroxyquinazoline was a gift from Dr. John B. Hynes of this university. Tetrahydrofolate was prepared by the method of Hat& et al. 15) and stored under argon. Concentration of tetrahydrofolate was determined using a molar extinction coeffkient at 298 nm of 22,000. Stability was measured using the reactivity of the formylated derivative with thymidylate synthetase. Molar extinction coefficients for methylene tetrahydrofolate and dihydrofolate of 32,000 at 294 nm and 6400 at 340 nm, respectively, were used. The thymidylate synthetase was a gift from Dr. D. V. Santi, University of California, San Francisco. used:
CM-,
carboxymethyl.
306
JONES
AND
Polyacrylamide disc gel electrophoresis of the native enzyme was conducted using the method of Ornstein (6) and Davis (7), and in the presence of sodium dodecyl sulfate by the method of Weber and Osborn (8). Protein was typically stained with Coomassie brilliant blue R. A Beckman Acta CIII uv-visible spectrophotometer was used in the double beam mode for these studies. The thermostated cell chamber was maintained at 25°C by circulating water. Routine assays for enzymic activity with P-phenylserine as substrate were carried out at 279 nm, measuring the rate of benzaldehyde appearance. A molar absorption difference between P-phenylserine and benzaldehyde of 1410 was used. Unless otherwise indicated, solution conditions for assays were 0.1 M DLthreo-P-phenylserine buffered to pH 7.5 with 0.01 M potassium phosphate in open cuvettes. The threonine aldolase reaction was measured with the aid of alcohol dehydrogenase (2). Serine transhydroxymethylase activity was determined by the method of Schixh and Gross (2). Protein concentrations were estimated by the method of Warburg and Christian (9). Equilibrium binding studies were conducted using a perturbation of the enzyme spectrum at 495 nm (IO). Enzyme p&ication. Bovine liver was obtained from freshly slaughtered beef and frozen immediately on Dry Ice. This was necessary to prevent loss of enzymic activity. Once frozen, the liver could be stored for several days at -70°C without appreciable loss of activity. Partial purification of the enzyme was accomplished by modifications of the procedures of Schirch and Mason (ll), Schirch and Gross (2) and Ulevitch (4) for the rabbit liver and lamb liver enzymes. All buffers, unless otherwise indicated, contain 2 mM p-mercaptoethanol. Three hundred grams of beef liver was homogenized in 750 ml of 0.01 M potassium phosphate buffer, pH 7.3, containing 1 rnM EDTA, and 0.1 rnM pyridoxal 5 phosphate. The homogenate was centrifuged at 16,300g for 20 min to remove cell debris. The supernatant was then heated to 55°C on a steam bath for 5 min and cooled immediately to 5°C by the direct addition of ice. Precipitated protein was removed by centrifugation at 16,300g for 20 min. To the supernatant was then added, with stirring, solid ammonium sulfate to a final concentration of 504, saturation, and stirring was continued for an additional 10 min. The mixture was centrifuged for 20 min at 16,300g and the pelleted protein containing the enzyme suspended in a minimum volume of 5 mM potassium phosphate buffer, pH 7.3, containing 1 mM EDTA and 0.1 mM pyridoxal 5.phosphate. The resuspended enzyme was then absorbed to three times its solution volume of CM-Sephadex previously equilibrated in the same buffer. The enzyme was eluted from the CM-Sephadex, in a Buchner funnel, by
PRIEST equilibration with 200 ml of 0.5 M potassium phosphate buffer, pH 7.7, containing 1 mM EDTA and 0.1 mM pyridoxal 5-phosphate. The eluted enzyme was precipitated at 60% saturation with solid ammonium sulfate, centrifuged for 20 min at 16,3OOg, and the pellet was dissolved in 5 mM potassium phosphate buffer and dialyzed overnight against this buffer. This dialysate was placed on a 1.0 x 15.0~cm column of the folate affinity gel material (to be discussed later). The column was washed with the above mentioned 5 ,mM potassium phosphate buffer, pH 7.3, until no further protein eluted (usually 50-75 ml). No serine transhydroxymethylase was eluted during washing. The enzyme could be eluted from the afRnity matrix using either 200 PM folate or a linear potassium phosphate gradient consisting of 100 ml of equilibrating buffer and 100 ml of 1.0 M potassium phosphate. A typical elution profile is shown in Fig. 1. The fractions containing the highest activity were pooled and dialyzed against 20 volumes of 0.01 M potassium phosphate buffer, pH 7.3. The specific activity of this preparation was 2.3 pmol of benzaldehyde min’ (mg of protein) I_ This is identical to the highest specific activity obtained for the enzyme from lamb liver (4). The ALX,,A ,B,, ratio obtained varied from 7.5 to 9.5 (2, 4, 10, 111. Polyacrylamide disc gel electrophoresis resulted in one band containing over 90% of the Coomassie bluestainable material. Companion gels were developed using a modification of the activity stain described by Ulevitch (4). The gels were incubated for 5 min at 37°C in a solution of 0.1 M nn-threo-P-phenylserine and then placed in a freshly prepared solution of 2 N phenylhydrazine at 37°C prepared by the addition of triethylamine to a water solution of phenylhydrazine hydrochloride. An opaque white band appeared after several minutes, corresponding to the Coomassie blue-stainable material. Prolonged storage of gels developed using this procedure resulted in yellowing, caused by decomposition of the phenylhy-
I
s s I
0
FIG. 1. Chromatography of serine transhydroxymethylase on a folate affinity column. Three-milliliter fractions were collected throughout.
SERINE
TRANSHYDROXYMETHYLASE
drazine. Sodium dodecyl sulfate-gel electrophoresis also yielded a single band. Extrapolation from a plot of log M, vs mobility, using standard proteins, gave a subunit molecular weight of approximately 50,000. Results of a typical purification are shown in Table I. It can be seen that the crude homogenate contained less total enzymatic activity than was present in the subsequent step. This inhibition was shown to be due to the presence of a heat-labile, nondialyzable, ammonium sulfate-precipitable component. Apparent activation also took place at the batch CM-Sephadex step. This could be attributed to restoration of a full complement of pyridoxal 5-phosphate. Preparataon. of affinity matrix. Hexanediamino Sepharose 4-B was prepared using the procedure of Weibel (12). Two hundred milliliters of Sepharose 4B was suspended in 50 ml of water, placed in an ice bath at o”C, and gently stirred. The pH was adjusted to 9-11 and a solution containing 60 g of cyanogen bromide in 60 ml of dimethylformamide added. The mixture was gently stirred and the pH maintained between 9 and 11 by intermittent addition of 20%, NaOH. After approximately 1 h the pH had stabilized. The suspension was stirred for an additional 2 h, after which 90 g of solid 1,6-hexanediamine was added. The suspension was then gently stirred overnight at 4°C. The hexanediamino Sepharose 4-B was washed with 4 liters of 1 M NaOH, followed by distilled water, ad resuspended in water for storage. Folate was coupled using a modification of the method of Slater et al. (13). To 100 ml of the gel was added a solution of 110 mg (0.25 mmol) of folic acid in 25 ml of 0.5 M NaHCO., and 25 ml of dimethylformamide. The pH of the resulting suspension was adjusted to 5.5 to 6.0 and 520 mg of I-ethyl-3-(3. dimethylaminopropyl)-carbodiimide hydrochloride added. The reaction mixture was maintained at room temperature with gentle stirring overnight, filtered, and washed with 1800 ml of 1 M NaCl followed by 1800 ml of distilled water. An absorbance reading on filtrates using epX.’ of 27,600 for folic acid, indicated approximately 1.5 pmol of folate bound per milliliter of gel. Unreacted amino groups were
capped by the addition of 250 mg of sodium acetate and 250 mg of the carbodiimide followed by gentle stirring overnight at pH 5.5. The capped affinity matrix was then washed with 1 liter of 1 M NaCl and 1 liter of distilled water and equilibrated in 5 mM potassium phosphate buffer, pH 7.3. If free amino groups were not capped, separation of serine transhydroxymethylase was not as sharp. RESULTS
It can be seen in Fig. 2 that the @-phenylserine aldolase reaction catalyzed by bovine serine transhydroxymethylase is stimulated by folate. Maximal stimulation occurs at 15-20 ELM and subsequently diminished. Tetrahydrofolate also stimulates the aldolase reaction in this concen-
2.01
__--.
Purification
Step
Volume (ml)
Homogenization Heat 50% Ammonium sulfate Batch CM-Sephadex 60% Ammonium sulfate Affinity chromatography
750 900 90 185 13 6
OF SERINE
Protein (mglml) 85.4 20 56.4 0.73 7.0 1.2
, 0
IO &igancj
,
,
20 pM
30
FIG. 2. Initial velocity as a function of folate (A), tetrahydrofolate ( x), and 2.amino-6-(4-carbethoxybenzylamino)+hydroxyquinazoline (0) concentration. Initial velocities were determined in the presence of 0.1 M DL-three-P-phenylserine and 0.01 M potassium phosphate, pH 7.5. In the case of folate and the quinazoline, reactions were initiated by the mixing of 2.3 ml of substrate and 0.2 ml 13.5 keg) of enzyme solution. A 3.0-ml total volume was used for tetrahydrofolate samples. Initial velocities are reported as micromoles of benzaldehyde produced per minute and have been normalized to 1 mg of enzyme.
TABLE PURIFICATION
307
ACTIVATION
I
TRANSHYDROXYMETHYLASE
Total protein (mg) 64,056 18,068 5,079 134 91.1 7.2
Activity per milliliter 0.11 0.15 1.1 0.83 6.6 2.8
-__--
Total activity
Specific activity
Yield (o/c)
79.8
0.0012 0.0074 0.0200 1.15 0.95 2.3
100 168 125 193 108 21
134.0 99.6 154.0 86.3 16.8
308
JONES
AND
tration range but does not exhibit a maximum. The threonine aldolase reaction catalyzed by serine transhydroxymethylase was not stimulated by folate in the same concentration range, and the serine transhydroxymethylase reaction was negligibly inhibited in agreement with Rowe and Lewis (14). Tetrahydrofolate could conceivably activate the p-phenylserine aldolase reaction by acting as either a substrate type of acceptor or a catalytic type of acceptor for the hydroxybenzyl group. Although not nearly as likely, folate could also activate in this manner. Figure 3 shows that folate does not act as a stoichiometric substrate. If this were the case, stimulation could only have occurred during the initial portions of the reaction. Were folate participating stoichiometrically, it would have been exhausted prior to the time indicated by the arrow in Fig. 3, and the rate would have returned to that of the non-folate-stimulated reaction. In addition, uv scans of the folate spectra were identical before and after the stimulated reaction. Were folate acting as an acceptor, the product which would have to be formed would almost certainly be highly unstable. Thus, the possibility that oxidized folate could act as a transient acceptor was also investigated. Again, this function for folate activation is unlikely. A quinazoline derivative of isofolate, compound II, is also an activator of the P-phenylserine aldolase reaction (see Fig. 2). It can be seen that the structural characteristics of this analog are similar to
I, OH
H
I coo0
those of folate, Compound I, but it is a much less likely acceptor for the hydroxybenzyl group. Figure 4 shows that the pH dependence of the p-phenylserine aldolase activity is
PRIEST
0.15 -
A 279
0.10 -
Time
(min)
3. Time course of P-phenylserine aldolase reaction in the presence and absence of folate. The upper line shows the time course in the presence of 17 ELM folate. The substrate for both was 0.1 M pphenylserine in 0.01 M potassium phosphate buffer, pH 7.5. Suppression was used to start both curves at the same position on the absorbance scale. The arrow indicates the theoretical time required to exhaust folate if it were acting as a substrate. FIG.
3.0 -
.>
2.0 -
1.0 -
'6.0
I
70
8.0 PH
FIG. 4. pH dependence of P-phenylserine aldolase activity in the presence of 14 pM folate (Al and with no folate present (0). Reactions were initiated by the addition of 20 pg of enzyme to 3.0 ml cells containing 0.1 M nL-thero-p-phenylserine and 0.01 M potassium phosphate. Error bars represent standard deviations for four separate determinations.
affected by the presence of folate. Potassium phosphate was the only buffer used to prevent additional complications from other specific buffer-ions effects. The pHrate profile in the absence of folate increases gradually up to approximately pH 7.8 and then declines sharply. The folateactivated profile is qualitatively similar but with the break occurring approximately 0.4 pH unit lower. Since potassium phosphate, as well as other salts, have previously been reported to inhibit rabbit liver serine transhydroxymethylase (3) and results with the affinity column (see Fig. 1) indicated that potas-
SERINE
TRANSHYDROXYMETHYLASE
sium phosphate was capable of causing dissociation of the enzyme from immobilized folate, the effects of this component on the folate activation were investigated. Figure 5 shows a plot of the dependence of velocity on phosphate ion concentration in the presence and absence of folate. It can be seen that activation by folate is much more pronounced at lower potassium phosphate concentrations. Both potassium chloride and potassium sulfate behaved in a qualitatively similar manner. The direct binding of folate to beef liver serine transhydroxymethylase was further investigated using methods described by Schirch and Ropp (10) for tetrahydrofolate binding to the rabbit liver enzyme. Using perturbation of absorbance at 495 nm, dissociation constants of 8.3 x 1OV’ M for the enzyme-folate complex and 5.6 x lo-” M for the enzyme-tetrahydrofolate complex were determined. When the rate of the aldolase reaction was determined over a range of p-phenylserine concentrations, approximately linear reciprocal plots were obtained (see Fig. 6). As folata concentrations increase, slopes of these reciprocal plots are significantly diminished but intercepts remain essentially unchanged up to 10 ,UM folate. At higher folate concentrations (e.g., 50 PM), on the other hand, the intercept is increased. While the reciprocal plots themselves are apparently linear in the presence of folate, replots of slopes and intercepts are clearly nonlinear when plotted either as a direct or a reciprocal function of folate.
OOh [Potassium
Phosphate]
(M)
FIG. 5. Initial velocity of the o-phenylserine alreaction as a function of potassium phosphate concentration in the presence of 14 FM folate (0) and
dolase
with
no folate
present
(A).
ACTIVATION
FIG. 6. Reciprocal plots for p-phenylserine ate concentrations shown. Reaction conditions the same as in Fig. 2.
309
at folwere
DISCUSSION
Chen and Schirch (15), working with the rabbit liver enzyme, have reported that tetrahydrofolate stimulates the conversion of glycine and formaldehyde to serine by acting as an acceptor or carrier of the formaldehyde group. We observe a stimulatory effect of tetrahydrofolate, folate, and a quinazoline derivative of isofolate on the p-phenylserine aldolase reaction catalyzed by the beef liver enzyme. The latter two compounds appear unlikely as even transient acceptors for the aldehydic group because of the very different oxidation states and, in the case of the quinazoline, the absence of N” and NJ” nitrogens. Schirch and Ropp (10) have measured the affinity of tetrahydrofolate for the rabbit liver enzyme (Kcli, = 7.7 x 10m5M). We observe a similar value for the beef liver enzyme. Further, folate binds with essentially the same affinity as tetrahydrofolate. The affinity column results (Fig. 1) were possible because folate can bind directly to the free enzyme. Phosphate ion, which can be used to elute the enzyme from the affnity matrix, diminishes activation by folate. This is consistent with decreased folate binding at high phosphate ion concentration suggested by kinetic experiments. The altered pH-rate profile in the presence of folate suggests that an ionizable group on the enzyme participates in substrate binding or reactivity because folate does not contain a functional group typically capable of ionization in this pH region. Folate exhibits a stimulatory effect at low concentrations which is diminished at
310
JONES
AND
higher concentrations. These effects can be seen on intercepts of reciprocal plots with fl-phenylserine. These kinetic data are consistent with a mechanism in which folate binds to the free enzyme, enhances pphenylserine binding, and subsequently dissociates. The dissociation step occurs prior to total product loss. Reciprocal plots for P-phenylserine are approximately linear in the absence of folate over the concentration range that could be experimentally observed. These data are consistent with a model requiring only a single enzyme-substrate complex. There are at least two products evolved during turnover, benzaldehyde and glytine. No attempt has been made to ascertain the order (or lack of order) of product release. When reciprocal plots of p-phenylserine are prepared at various folate concentrations, both slope and intercept effects can be detected. However, intercept effects are negligible at low folate concentrations. On the other hand, at high folate concentrations intercepts are increased. Slopes of the reciprocal plots are decreased throughout the concentration range of folate used but approach a nonzero value. The absence of intercept effects by folate at low concentrations suggests that under these conditions the predominant reciprocal rate equation terms containing folate will also contain @-phenylserine concentration. This may be viewed as analogous to rate equation terms for “competitive” inhibitors. Of course, such an analogy is only an approximation and, while it may aid in understanding the overall proposed mechanism, the ligand, folate, is an activator and not an inhibitor. Therefore, true “dead end” behavior is not possible. Rather, it is proposed that folate can bind “competitively” with p-phenylserine for the free enzyme but, once folate is bound, the new enzyme form can bind p-phenylserine with greater facility. This model would allow stimulation of the reaction by providing another, faster pathway for the binding of P-phenylserine. It can be seen in Fig. 2 and in the reciprocal plots of Fig. 6 that the folate concentration dependence exhibits a maximum. Therefore, as high folate concentrations
PRIEST
are obtained, the reaction is slowed. These results suggest that there is a point in the reaction pathway in which the presence of folate is inhibitory. Such a step is obtained if folate is released prior to product release. This requirement may or may not be absolute. It is difficult to obtain experimental data sufficient to extrapolate accurately to infinite folate concentration. A model summarizing the proposed mechanism is shown in Fig. 7. The full reciprocal rate equation for this model using the symbolism of Fisher and co-workers (16, 17) is shown below.
[II k,
k:,
ksh,
k;,k,,k,
where F is folate, C#Jis P-phenylserine, and u’ is initial velocity per mole of enzyme. It can be seen that this equation not only predicts the observed nonlinear slope and intercept dependence of reciprocal plots but also predicts that the reciprocal plots themselves will be nonlinear. Linearity can be obtained with an assumption regarding one rate constant. That is, if the “on” constant for the binding of folate to the free enzyme, h;,, is high relative to the P-phenylserine constant, h ,, then the pphenylserine dependence of the fractional velocity terms, f, becomes negligible. This assumption gains credence when the relative concentrations of folate and P-phenyl-
$I Ser FIG. 7. Proposed the fi-phenylserine
PI ‘P2 mechanism for the activation aldolase reaction by folate.
of
SERINE
TRANSHYDROXYMETHYLASE
serine used for these studies are compared. This assumption is not imperative to the argument, because it is notoriously diffcult to prove that a particular set of data points is, indeed, linear. Slight to moderate nonlinearity could be present and experimentally unobservable. The proposed model suggests that the predominant role of folate is to facilitate pphenylserine binding. It can also be seen in Fig. 7, however, that folate binds to an enzyme form other than the free enzyme. Such binding is nonproductive and is even ultimately inhibitory. Inhibitory (numerator) terms in the reciprocal rate equation will become much more significant at high folate concentrations and will ultimately dominate the equation (see Eq. [2]). 1 (‘
k / F k,,k ( F k, k: ‘- h,,k, k[d,
high
folate.
ACTIVATION
binding of the substrate. It is then lost prior to subsequent events in the overall reaction pathway. Enzymes requiring the binding of two substrates could utilize an analog of the second substrate to enhance binding of the first, followed by loss of the analog prior to the second substrate binding as suggested by Griffin and Brand (18) and Wells and Fisher (19). Such mechanisms of activation must be at least as feasible as remote site effector binding which must give rise to a more favorable site for substrate binding at a catalytic site by acting over extensive distances. ACKNOWLEDGMENTS Financial assistance was provided in part the 1973 South Carolina State Appropriation Research and the College of Dental Medicine.
121
The inability of high folate concentrations to eliminate ,&phenylserine dependence completely (i.e., slopes of p-phenylserine reciprocal plots do not approach zero) means that the k-&,.F/k,,k,.k.& term in the rabe eq$ation remains finite relative to the k.,.F/k,.ky term even at saturating folate concentrations. Thus, k-,,lk,, cannot be extremely small. If k,, is large (facilitated p-phenylserine binding to EF) then k ,, must also be large. Further, apparently folate cannot totally inhibit turnover of the enzyme. Thus, the relationship h,, 1 /z ,. must hold. This is consistent with activation by folate at low concentrations, which diminishes at higher concentrations. It is presumed that folate binding to the beef lives serine transhydroxyrnethylase occurs at the tetrahydrofolate-binding site. Thus, for aldolase reactions it is unnecessary for folate to exert a stimulatory influence through binding to a remote site, be it catalytic or regulatory. If indeed folate is exerting its influence primarily through action at the catalytic site, this system may provide a generally applicable example of substrate analog activation occurring due to direct interaction with a functional catalytic site. Such activation is often interpreted t,hrough the use of remote site models. In the present case, the activator is bound first, enhancing the
311
from for
REFERENCES 1. BLAKLEY, 2. SCHIRCH, Chcrn. 3. SCHIRCH, Chem. 4. ULEVITCII, versity 5. HATEFI,
6. 7. 8. 9. 10. 11. 12. 13. 14.
R. I,. (1955) Brochem. J. 61, 315. L., END GROSS, ‘1‘. (1968) J. Viol. 213, 5651. L., AND DILLER, A. (1971) J. Ricll. 246, 3961. R. J. (1971) Ph.D. Dissertation, Uniof Pennsylvania. Philadelphia. Y., TALBERT. D., OSBORN, M. J., AND HUENNEKENS, F. M. (1960) Biochem. Prep. 7, 89. ORNSTEIN, I,. (1964) Ann. N.Y. Acud. Sci. 121. 321. DAVIS, B. ,J. (1964) Ann. N.Y. Acad. Sci. 121, 404. WEBER, K., AND OSRORN, M. (1969) J. Riol. C’hpm. 214, 4406. WARBURG, 0.. Am CHRISTIAN, W. (1941) Riochern. z. 410, 384. SCHIRCH, L., AND ROPP, M. (1967) Biochemistry 6. 253. SCHIRCH, I,. G., AND MASON, M. (1963) J. Rlol. (:hern. 278 1032. c WEIBEL, h;. k. (19741 personal communication. SLATER, D. N., FORD, J. E., SCOTT, K. J., AND ANDREWS, P. (1972) FEBS L&t. 20, 302. ROWE, P. B., AND LEWIS, G. P. 119?3)Biochemistrv
12,
1962.
15. CHEN, M. S., AND SCHIRCH, I,. (19731 J. Riol. ChPrn. 248, 7979. 16. FISHER, J. R., AND HOAGLAND, V. D. (1968) Adtan. Biol. Med. Phvs. 12, 165. 17. FISHER, J. R., PRIEST, D. G., AND BARTON, J. R. (1972) J. Z’heoret. Biol. 37, 335. IU. GRIFFIN, C. C., AND BRAND, I,. (1968) Anh. Bwthem. Bio,ohys. 126, 856. 19. WELLS, B. D., AND FISHER, J. R. (1975) personal communication.
OF
BIOCHEMISTRY
AND
174,
BIOPHYSICS
Serine Transhydroxymethylase: Activation CLIFTON Department
of Biochemistry,
Mechanism by Folate
W. JONES,
Medical
University Received
(1976)
305-311
III, of
South
September
AND
of Aldolase
D. G. PRIEST
Carolina,
Charleston,
South
Carolina
29401
29, 1975
Affinity chromatographic methods have been developed to purify beef liver serine transhydroxymethylase. This enzyme catalyzes both the transfer of aldehydic groups from /3-hydroxy amino acids to tetrahydrofolate and the cleavage of p-hydroxy amino acids yielding the free aldehyde. Tetrahydrofolate, folate, and a quinazoline analog of isofolate were found to be a$.ivators of the p-phenylserine aldolase reaction catalyzed by serine transhydroxymethylase. Activation by folate was maxima1 at 20 FM, and higher concentrations diminished the activation. Evidence is presented suggesting that folate does not activate by providing an acceptor site for the aldehydic groups. Equilibrium binding studies showed that folate and tetrahydrofolate can bind the enzyme with essentially the same affinity. Double-reciprocal plots with P-phenylserine from steadystate kinetic experiments did not yield a l/u, intercept effect except at high folate concentrations. A mechanism is proposed in which folate binds readily to the enzymic active site, facilitating p-phenylserine binding. Folate is subsequently lost, at least partially, prior to product release and complete enzymic turnover.
Serine tran+ydroxymethyIase, L-serine:tetrahydrofolate 5, lo-hydroxymethyltransferase (EC 2.1.2.11, catalyzes the transfer of a hydroxymethyl group from serine to tetrahydrofolate (1) and also the cleavage of serine and other /3-hydroxy amino acids yielding glycine and the corresponding aldehydic product in an aldolasetype reaction (2). Several such P-hydroxy amino acids are substrates for the enzymes isolated from rabbit liver (3) and lamb liver (4). We have found folate, tetrahydrofolate, and a quinazoline analog of isofolate to be activators of the /3-phenylserine aldolase reaction catalyzed by bovine liver serine transhydroxymethylase. The aldolase reaction with threonine as substrate was not enhanced by folate under the conditions used. The relative contribution of folate to the structural suitability of the enzymic active site, as opposed to its participation as an acceptor for the aldehydic group, was investigated. Results of these studies are reported here and interpreted in terms of an enzymic mechanism of folate activation.
MATERIALS
’ Abbreviation 305
Copyright All rights
5: 1976 by Academic Press, Inc. of reproduction in any form reserved.
AND
METHODS
The EDTA, folic acid, nL-threo-P-phenylserine, Lserine, L-threonine, Coomassie brilliant blue R, pyridoxal5-phosphate, phenylhydrazine hydrochloride, and alcohol dehydrogenase were obtained from Sigma Chemical Company. Sepharose 4-B and CMSephadex’ were obtained from Pharmacia Fine Chemicals Company. Potassium phosphate, 1,6hexanediamine, sodium acetate, and dimethylformamide were obtained from Fisher Scientific Company. Ammonium sulfate was obtained from SchwarziMann. The I-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride was obtained from Pierce Chemical Company. The 2-amino-6-(4carbethoxybenzylamino)-4-hydroxyquinazoline was a gift from Dr. John B. Hynes of this university. Tetrahydrofolate was prepared by the method of Hat& et al. 15) and stored under argon. Concentration of tetrahydrofolate was determined using a molar extinction coeffkient at 298 nm of 22,000. Stability was measured using the reactivity of the formylated derivative with thymidylate synthetase. Molar extinction coefficients for methylene tetrahydrofolate and dihydrofolate of 32,000 at 294 nm and 6400 at 340 nm, respectively, were used. The thymidylate synthetase was a gift from Dr. D. V. Santi, University of California, San Francisco. used:
CM-,
carboxymethyl.
306
JONES
AND
Polyacrylamide disc gel electrophoresis of the native enzyme was conducted using the method of Ornstein (6) and Davis (7), and in the presence of sodium dodecyl sulfate by the method of Weber and Osborn (8). Protein was typically stained with Coomassie brilliant blue R. A Beckman Acta CIII uv-visible spectrophotometer was used in the double beam mode for these studies. The thermostated cell chamber was maintained at 25°C by circulating water. Routine assays for enzymic activity with P-phenylserine as substrate were carried out at 279 nm, measuring the rate of benzaldehyde appearance. A molar absorption difference between P-phenylserine and benzaldehyde of 1410 was used. Unless otherwise indicated, solution conditions for assays were 0.1 M DLthreo-P-phenylserine buffered to pH 7.5 with 0.01 M potassium phosphate in open cuvettes. The threonine aldolase reaction was measured with the aid of alcohol dehydrogenase (2). Serine transhydroxymethylase activity was determined by the method of Schixh and Gross (2). Protein concentrations were estimated by the method of Warburg and Christian (9). Equilibrium binding studies were conducted using a perturbation of the enzyme spectrum at 495 nm (IO). Enzyme p&ication. Bovine liver was obtained from freshly slaughtered beef and frozen immediately on Dry Ice. This was necessary to prevent loss of enzymic activity. Once frozen, the liver could be stored for several days at -70°C without appreciable loss of activity. Partial purification of the enzyme was accomplished by modifications of the procedures of Schirch and Mason (ll), Schirch and Gross (2) and Ulevitch (4) for the rabbit liver and lamb liver enzymes. All buffers, unless otherwise indicated, contain 2 mM p-mercaptoethanol. Three hundred grams of beef liver was homogenized in 750 ml of 0.01 M potassium phosphate buffer, pH 7.3, containing 1 rnM EDTA, and 0.1 rnM pyridoxal 5 phosphate. The homogenate was centrifuged at 16,300g for 20 min to remove cell debris. The supernatant was then heated to 55°C on a steam bath for 5 min and cooled immediately to 5°C by the direct addition of ice. Precipitated protein was removed by centrifugation at 16,300g for 20 min. To the supernatant was then added, with stirring, solid ammonium sulfate to a final concentration of 504, saturation, and stirring was continued for an additional 10 min. The mixture was centrifuged for 20 min at 16,300g and the pelleted protein containing the enzyme suspended in a minimum volume of 5 mM potassium phosphate buffer, pH 7.3, containing 1 mM EDTA and 0.1 mM pyridoxal 5.phosphate. The resuspended enzyme was then absorbed to three times its solution volume of CM-Sephadex previously equilibrated in the same buffer. The enzyme was eluted from the CM-Sephadex, in a Buchner funnel, by
PRIEST equilibration with 200 ml of 0.5 M potassium phosphate buffer, pH 7.7, containing 1 mM EDTA and 0.1 mM pyridoxal 5-phosphate. The eluted enzyme was precipitated at 60% saturation with solid ammonium sulfate, centrifuged for 20 min at 16,3OOg, and the pellet was dissolved in 5 mM potassium phosphate buffer and dialyzed overnight against this buffer. This dialysate was placed on a 1.0 x 15.0~cm column of the folate affinity gel material (to be discussed later). The column was washed with the above mentioned 5 ,mM potassium phosphate buffer, pH 7.3, until no further protein eluted (usually 50-75 ml). No serine transhydroxymethylase was eluted during washing. The enzyme could be eluted from the afRnity matrix using either 200 PM folate or a linear potassium phosphate gradient consisting of 100 ml of equilibrating buffer and 100 ml of 1.0 M potassium phosphate. A typical elution profile is shown in Fig. 1. The fractions containing the highest activity were pooled and dialyzed against 20 volumes of 0.01 M potassium phosphate buffer, pH 7.3. The specific activity of this preparation was 2.3 pmol of benzaldehyde min’ (mg of protein) I_ This is identical to the highest specific activity obtained for the enzyme from lamb liver (4). The ALX,,A ,B,, ratio obtained varied from 7.5 to 9.5 (2, 4, 10, 111. Polyacrylamide disc gel electrophoresis resulted in one band containing over 90% of the Coomassie bluestainable material. Companion gels were developed using a modification of the activity stain described by Ulevitch (4). The gels were incubated for 5 min at 37°C in a solution of 0.1 M nn-threo-P-phenylserine and then placed in a freshly prepared solution of 2 N phenylhydrazine at 37°C prepared by the addition of triethylamine to a water solution of phenylhydrazine hydrochloride. An opaque white band appeared after several minutes, corresponding to the Coomassie blue-stainable material. Prolonged storage of gels developed using this procedure resulted in yellowing, caused by decomposition of the phenylhy-
I
s s I
0
FIG. 1. Chromatography of serine transhydroxymethylase on a folate affinity column. Three-milliliter fractions were collected throughout.
SERINE
TRANSHYDROXYMETHYLASE
drazine. Sodium dodecyl sulfate-gel electrophoresis also yielded a single band. Extrapolation from a plot of log M, vs mobility, using standard proteins, gave a subunit molecular weight of approximately 50,000. Results of a typical purification are shown in Table I. It can be seen that the crude homogenate contained less total enzymatic activity than was present in the subsequent step. This inhibition was shown to be due to the presence of a heat-labile, nondialyzable, ammonium sulfate-precipitable component. Apparent activation also took place at the batch CM-Sephadex step. This could be attributed to restoration of a full complement of pyridoxal 5-phosphate. Preparataon. of affinity matrix. Hexanediamino Sepharose 4-B was prepared using the procedure of Weibel (12). Two hundred milliliters of Sepharose 4B was suspended in 50 ml of water, placed in an ice bath at o”C, and gently stirred. The pH was adjusted to 9-11 and a solution containing 60 g of cyanogen bromide in 60 ml of dimethylformamide added. The mixture was gently stirred and the pH maintained between 9 and 11 by intermittent addition of 20%, NaOH. After approximately 1 h the pH had stabilized. The suspension was stirred for an additional 2 h, after which 90 g of solid 1,6-hexanediamine was added. The suspension was then gently stirred overnight at 4°C. The hexanediamino Sepharose 4-B was washed with 4 liters of 1 M NaOH, followed by distilled water, ad resuspended in water for storage. Folate was coupled using a modification of the method of Slater et al. (13). To 100 ml of the gel was added a solution of 110 mg (0.25 mmol) of folic acid in 25 ml of 0.5 M NaHCO., and 25 ml of dimethylformamide. The pH of the resulting suspension was adjusted to 5.5 to 6.0 and 520 mg of I-ethyl-3-(3. dimethylaminopropyl)-carbodiimide hydrochloride added. The reaction mixture was maintained at room temperature with gentle stirring overnight, filtered, and washed with 1800 ml of 1 M NaCl followed by 1800 ml of distilled water. An absorbance reading on filtrates using epX.’ of 27,600 for folic acid, indicated approximately 1.5 pmol of folate bound per milliliter of gel. Unreacted amino groups were
capped by the addition of 250 mg of sodium acetate and 250 mg of the carbodiimide followed by gentle stirring overnight at pH 5.5. The capped affinity matrix was then washed with 1 liter of 1 M NaCl and 1 liter of distilled water and equilibrated in 5 mM potassium phosphate buffer, pH 7.3. If free amino groups were not capped, separation of serine transhydroxymethylase was not as sharp. RESULTS
It can be seen in Fig. 2 that the @-phenylserine aldolase reaction catalyzed by bovine serine transhydroxymethylase is stimulated by folate. Maximal stimulation occurs at 15-20 ELM and subsequently diminished. Tetrahydrofolate also stimulates the aldolase reaction in this concen-
2.01
__--.
Purification
Step
Volume (ml)
Homogenization Heat 50% Ammonium sulfate Batch CM-Sephadex 60% Ammonium sulfate Affinity chromatography
750 900 90 185 13 6
OF SERINE
Protein (mglml) 85.4 20 56.4 0.73 7.0 1.2
, 0
IO &igancj
,
,
20 pM
30
FIG. 2. Initial velocity as a function of folate (A), tetrahydrofolate ( x), and 2.amino-6-(4-carbethoxybenzylamino)+hydroxyquinazoline (0) concentration. Initial velocities were determined in the presence of 0.1 M DL-three-P-phenylserine and 0.01 M potassium phosphate, pH 7.5. In the case of folate and the quinazoline, reactions were initiated by the mixing of 2.3 ml of substrate and 0.2 ml 13.5 keg) of enzyme solution. A 3.0-ml total volume was used for tetrahydrofolate samples. Initial velocities are reported as micromoles of benzaldehyde produced per minute and have been normalized to 1 mg of enzyme.
TABLE PURIFICATION
307
ACTIVATION
I
TRANSHYDROXYMETHYLASE
Total protein (mg) 64,056 18,068 5,079 134 91.1 7.2
Activity per milliliter 0.11 0.15 1.1 0.83 6.6 2.8
-__--
Total activity
Specific activity
Yield (o/c)
79.8
0.0012 0.0074 0.0200 1.15 0.95 2.3
100 168 125 193 108 21
134.0 99.6 154.0 86.3 16.8
308
JONES
AND
tration range but does not exhibit a maximum. The threonine aldolase reaction catalyzed by serine transhydroxymethylase was not stimulated by folate in the same concentration range, and the serine transhydroxymethylase reaction was negligibly inhibited in agreement with Rowe and Lewis (14). Tetrahydrofolate could conceivably activate the p-phenylserine aldolase reaction by acting as either a substrate type of acceptor or a catalytic type of acceptor for the hydroxybenzyl group. Although not nearly as likely, folate could also activate in this manner. Figure 3 shows that folate does not act as a stoichiometric substrate. If this were the case, stimulation could only have occurred during the initial portions of the reaction. Were folate participating stoichiometrically, it would have been exhausted prior to the time indicated by the arrow in Fig. 3, and the rate would have returned to that of the non-folate-stimulated reaction. In addition, uv scans of the folate spectra were identical before and after the stimulated reaction. Were folate acting as an acceptor, the product which would have to be formed would almost certainly be highly unstable. Thus, the possibility that oxidized folate could act as a transient acceptor was also investigated. Again, this function for folate activation is unlikely. A quinazoline derivative of isofolate, compound II, is also an activator of the P-phenylserine aldolase reaction (see Fig. 2). It can be seen that the structural characteristics of this analog are similar to
I, OH
H
I coo0
those of folate, Compound I, but it is a much less likely acceptor for the hydroxybenzyl group. Figure 4 shows that the pH dependence of the p-phenylserine aldolase activity is
PRIEST
0.15 -
A 279
0.10 -
Time
(min)
3. Time course of P-phenylserine aldolase reaction in the presence and absence of folate. The upper line shows the time course in the presence of 17 ELM folate. The substrate for both was 0.1 M pphenylserine in 0.01 M potassium phosphate buffer, pH 7.5. Suppression was used to start both curves at the same position on the absorbance scale. The arrow indicates the theoretical time required to exhaust folate if it were acting as a substrate. FIG.
3.0 -
.>
2.0 -
1.0 -
'6.0
I
70
8.0 PH
FIG. 4. pH dependence of P-phenylserine aldolase activity in the presence of 14 pM folate (Al and with no folate present (0). Reactions were initiated by the addition of 20 pg of enzyme to 3.0 ml cells containing 0.1 M nL-thero-p-phenylserine and 0.01 M potassium phosphate. Error bars represent standard deviations for four separate determinations.
affected by the presence of folate. Potassium phosphate was the only buffer used to prevent additional complications from other specific buffer-ions effects. The pHrate profile in the absence of folate increases gradually up to approximately pH 7.8 and then declines sharply. The folateactivated profile is qualitatively similar but with the break occurring approximately 0.4 pH unit lower. Since potassium phosphate, as well as other salts, have previously been reported to inhibit rabbit liver serine transhydroxymethylase (3) and results with the affinity column (see Fig. 1) indicated that potas-
SERINE
TRANSHYDROXYMETHYLASE
sium phosphate was capable of causing dissociation of the enzyme from immobilized folate, the effects of this component on the folate activation were investigated. Figure 5 shows a plot of the dependence of velocity on phosphate ion concentration in the presence and absence of folate. It can be seen that activation by folate is much more pronounced at lower potassium phosphate concentrations. Both potassium chloride and potassium sulfate behaved in a qualitatively similar manner. The direct binding of folate to beef liver serine transhydroxymethylase was further investigated using methods described by Schirch and Ropp (10) for tetrahydrofolate binding to the rabbit liver enzyme. Using perturbation of absorbance at 495 nm, dissociation constants of 8.3 x 1OV’ M for the enzyme-folate complex and 5.6 x lo-” M for the enzyme-tetrahydrofolate complex were determined. When the rate of the aldolase reaction was determined over a range of p-phenylserine concentrations, approximately linear reciprocal plots were obtained (see Fig. 6). As folata concentrations increase, slopes of these reciprocal plots are significantly diminished but intercepts remain essentially unchanged up to 10 ,UM folate. At higher folate concentrations (e.g., 50 PM), on the other hand, the intercept is increased. While the reciprocal plots themselves are apparently linear in the presence of folate, replots of slopes and intercepts are clearly nonlinear when plotted either as a direct or a reciprocal function of folate.
OOh [Potassium
Phosphate]
(M)
FIG. 5. Initial velocity of the o-phenylserine alreaction as a function of potassium phosphate concentration in the presence of 14 FM folate (0) and
dolase
with
no folate
present
(A).
ACTIVATION
FIG. 6. Reciprocal plots for p-phenylserine ate concentrations shown. Reaction conditions the same as in Fig. 2.
309
at folwere
DISCUSSION
Chen and Schirch (15), working with the rabbit liver enzyme, have reported that tetrahydrofolate stimulates the conversion of glycine and formaldehyde to serine by acting as an acceptor or carrier of the formaldehyde group. We observe a stimulatory effect of tetrahydrofolate, folate, and a quinazoline derivative of isofolate on the p-phenylserine aldolase reaction catalyzed by the beef liver enzyme. The latter two compounds appear unlikely as even transient acceptors for the aldehydic group because of the very different oxidation states and, in the case of the quinazoline, the absence of N” and NJ” nitrogens. Schirch and Ropp (10) have measured the affinity of tetrahydrofolate for the rabbit liver enzyme (Kcli, = 7.7 x 10m5M). We observe a similar value for the beef liver enzyme. Further, folate binds with essentially the same affinity as tetrahydrofolate. The affinity column results (Fig. 1) were possible because folate can bind directly to the free enzyme. Phosphate ion, which can be used to elute the enzyme from the affnity matrix, diminishes activation by folate. This is consistent with decreased folate binding at high phosphate ion concentration suggested by kinetic experiments. The altered pH-rate profile in the presence of folate suggests that an ionizable group on the enzyme participates in substrate binding or reactivity because folate does not contain a functional group typically capable of ionization in this pH region. Folate exhibits a stimulatory effect at low concentrations which is diminished at
310
JONES
AND
higher concentrations. These effects can be seen on intercepts of reciprocal plots with fl-phenylserine. These kinetic data are consistent with a mechanism in which folate binds to the free enzyme, enhances pphenylserine binding, and subsequently dissociates. The dissociation step occurs prior to total product loss. Reciprocal plots for P-phenylserine are approximately linear in the absence of folate over the concentration range that could be experimentally observed. These data are consistent with a model requiring only a single enzyme-substrate complex. There are at least two products evolved during turnover, benzaldehyde and glytine. No attempt has been made to ascertain the order (or lack of order) of product release. When reciprocal plots of p-phenylserine are prepared at various folate concentrations, both slope and intercept effects can be detected. However, intercept effects are negligible at low folate concentrations. On the other hand, at high folate concentrations intercepts are increased. Slopes of the reciprocal plots are decreased throughout the concentration range of folate used but approach a nonzero value. The absence of intercept effects by folate at low concentrations suggests that under these conditions the predominant reciprocal rate equation terms containing folate will also contain @-phenylserine concentration. This may be viewed as analogous to rate equation terms for “competitive” inhibitors. Of course, such an analogy is only an approximation and, while it may aid in understanding the overall proposed mechanism, the ligand, folate, is an activator and not an inhibitor. Therefore, true “dead end” behavior is not possible. Rather, it is proposed that folate can bind “competitively” with p-phenylserine for the free enzyme but, once folate is bound, the new enzyme form can bind p-phenylserine with greater facility. This model would allow stimulation of the reaction by providing another, faster pathway for the binding of P-phenylserine. It can be seen in Fig. 2 and in the reciprocal plots of Fig. 6 that the folate concentration dependence exhibits a maximum. Therefore, as high folate concentrations
PRIEST
are obtained, the reaction is slowed. These results suggest that there is a point in the reaction pathway in which the presence of folate is inhibitory. Such a step is obtained if folate is released prior to product release. This requirement may or may not be absolute. It is difficult to obtain experimental data sufficient to extrapolate accurately to infinite folate concentration. A model summarizing the proposed mechanism is shown in Fig. 7. The full reciprocal rate equation for this model using the symbolism of Fisher and co-workers (16, 17) is shown below.
[II k,
k:,
ksh,
k;,k,,k,
where F is folate, C#Jis P-phenylserine, and u’ is initial velocity per mole of enzyme. It can be seen that this equation not only predicts the observed nonlinear slope and intercept dependence of reciprocal plots but also predicts that the reciprocal plots themselves will be nonlinear. Linearity can be obtained with an assumption regarding one rate constant. That is, if the “on” constant for the binding of folate to the free enzyme, h;,, is high relative to the P-phenylserine constant, h ,, then the pphenylserine dependence of the fractional velocity terms, f, becomes negligible. This assumption gains credence when the relative concentrations of folate and P-phenyl-
$I Ser FIG. 7. Proposed the fi-phenylserine
PI ‘P2 mechanism for the activation aldolase reaction by folate.
of
SERINE
TRANSHYDROXYMETHYLASE
serine used for these studies are compared. This assumption is not imperative to the argument, because it is notoriously diffcult to prove that a particular set of data points is, indeed, linear. Slight to moderate nonlinearity could be present and experimentally unobservable. The proposed model suggests that the predominant role of folate is to facilitate pphenylserine binding. It can also be seen in Fig. 7, however, that folate binds to an enzyme form other than the free enzyme. Such binding is nonproductive and is even ultimately inhibitory. Inhibitory (numerator) terms in the reciprocal rate equation will become much more significant at high folate concentrations and will ultimately dominate the equation (see Eq. [2]). 1 (‘
k / F k,,k ( F k, k: ‘- h,,k, k[d,
high
folate.
ACTIVATION
binding of the substrate. It is then lost prior to subsequent events in the overall reaction pathway. Enzymes requiring the binding of two substrates could utilize an analog of the second substrate to enhance binding of the first, followed by loss of the analog prior to the second substrate binding as suggested by Griffin and Brand (18) and Wells and Fisher (19). Such mechanisms of activation must be at least as feasible as remote site effector binding which must give rise to a more favorable site for substrate binding at a catalytic site by acting over extensive distances. ACKNOWLEDGMENTS Financial assistance was provided in part the 1973 South Carolina State Appropriation Research and the College of Dental Medicine.
121
The inability of high folate concentrations to eliminate ,&phenylserine dependence completely (i.e., slopes of p-phenylserine reciprocal plots do not approach zero) means that the k-&,.F/k,,k,.k.& term in the rabe eq$ation remains finite relative to the k.,.F/k,.ky term even at saturating folate concentrations. Thus, k-,,lk,, cannot be extremely small. If k,, is large (facilitated p-phenylserine binding to EF) then k ,, must also be large. Further, apparently folate cannot totally inhibit turnover of the enzyme. Thus, the relationship h,, 1 /z ,. must hold. This is consistent with activation by folate at low concentrations, which diminishes at higher concentrations. It is presumed that folate binding to the beef lives serine transhydroxyrnethylase occurs at the tetrahydrofolate-binding site. Thus, for aldolase reactions it is unnecessary for folate to exert a stimulatory influence through binding to a remote site, be it catalytic or regulatory. If indeed folate is exerting its influence primarily through action at the catalytic site, this system may provide a generally applicable example of substrate analog activation occurring due to direct interaction with a functional catalytic site. Such activation is often interpreted t,hrough the use of remote site models. In the present case, the activator is bound first, enhancing the
311
from for
REFERENCES 1. BLAKLEY, 2. SCHIRCH, Chcrn. 3. SCHIRCH, Chem. 4. ULEVITCII, versity 5. HATEFI,
6. 7. 8. 9. 10. 11. 12. 13. 14.
R. I,. (1955) Brochem. J. 61, 315. L., END GROSS, ‘1‘. (1968) J. Viol. 213, 5651. L., AND DILLER, A. (1971) J. Ricll. 246, 3961. R. J. (1971) Ph.D. Dissertation, Uniof Pennsylvania. Philadelphia. Y., TALBERT. D., OSBORN, M. J., AND HUENNEKENS, F. M. (1960) Biochem. Prep. 7, 89. ORNSTEIN, I,. (1964) Ann. N.Y. Acud. Sci. 121. 321. DAVIS, B. ,J. (1964) Ann. N.Y. Acad. Sci. 121, 404. WEBER, K., AND OSRORN, M. (1969) J. Riol. C’hpm. 214, 4406. WARBURG, 0.. Am CHRISTIAN, W. (1941) Riochern. z. 410, 384. SCHIRCH, L., AND ROPP, M. (1967) Biochemistry 6. 253. SCHIRCH, I,. G., AND MASON, M. (1963) J. Rlol. (:hern. 278 1032. c WEIBEL, h;. k. (19741 personal communication. SLATER, D. N., FORD, J. E., SCOTT, K. J., AND ANDREWS, P. (1972) FEBS L&t. 20, 302. ROWE, P. B., AND LEWIS, G. P. 119?3)Biochemistrv
12,
1962.
15. CHEN, M. S., AND SCHIRCH, I,. (19731 J. Riol. ChPrn. 248, 7979. 16. FISHER, J. R., AND HOAGLAND, V. D. (1968) Adtan. Biol. Med. Phvs. 12, 165. 17. FISHER, J. R., PRIEST, D. G., AND BARTON, J. R. (1972) J. Z’heoret. Biol. 37, 335. IU. GRIFFIN, C. C., AND BRAND, I,. (1968) Anh. Bwthem. Bio,ohys. 126, 856. 19. WELLS, B. D., AND FISHER, J. R. (1975) personal communication.