Human Cytosolic and Mitochondrial Serine Hydroxymethyltransferase

Article Cite This: Biochemistry XXXX, XXX, XXX−XXX

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Human Cytosolic and Mitochondrial Serine Hydroxymethyltransferase Isoforms in Comparison: Full Kinetic Characterization and Substrate Inhibition Properties Angela Tramonti,†,‡ Caterina Nardella,‡ Martino L. di Salvo,‡ Anna Barile,‡ Francesca Cutruzzolà,*,‡ and Roberto Contestabile*,‡ †

Istituto di Biologia e Patologia Molecolari, Consiglio Nazionale delle Ricerche, Piazzale Aldo Moro 5, 00185 Roma, Italy Dipartimento di Scienze Biochimiche “A. Rossi Fanelli”, Sapienza Università di Roma, Laboratory affiliated to Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Piazzale Aldo Moro 5, 00185 Roma, Italy

Biochemistry Downloaded from pubs.acs.org by UNIV OF WINNIPEG on 12/07/18. For personal use only.

‡

ABSTRACT: Serine hydroxymethyltransferase (SHMT) catalyzes the reversible conversion of L-serine and tetrahydrofolate into glycine and 5,10-methylenetetrahydrofolate. This enzyme, which plays a pivotal role in one-carbon metabolism, is involved in cancer metabolic reprogramming and is a recognized target of chemotherapy intervention. In humans, two isoforms of the enzyme exist, which are commonly termed cytosolic SHMT1 and mitochondrial SHMT2. Considerable attention has been paid to the structural, mechanistic, and metabolic features of these isozymes. On the other hand, a detailed comparison of their catalytic and regulatory properties is missing, although this aspect seems to be considerably important, considering that SHMT1 and SHMT2 reside in different cellular compartments, where they play distinct roles in folate metabolism. Here we performed a full kinetic characterization of the serine hydroxymethyltransferase reaction catalyzed by SHMT1 and SHMT2, with a focus on pH dependence and substrate inhibition. Our investigation, which allowed the determination of all kinetic parameters of serine hydroxymethyltransferase forward and backward reactions, uncovered a previously unobserved substrate inhibition by L-serine and highlighted several interesting differences between SHMT1 and SHMT2. In particular, SHMT2 maintains a pronounced tetrahydrofolate substrate inhibition even at the alkaline pH characteristic of the mitochondrial matrix, whereas with SHMT1 this is almost abolished. At this pH, SHMT2 also shows a catalytic efficiency that is much higher than that of SHMT1. These observations suggest that such different properties represent an adaptation of the isoforms to the respective cellular environments and that substrate inhibition may be a form of regulation.

S

oligomeric state responds differently to the PLP cofactor.17 The reason for the existence of different SHMT isoforms, which have both been implicated in cancer,18−20 is related to the compartmentalization of one-carbon metabolism (Scheme 1). SHMT2 is localized in mitochondria, where it plays a crucial role in the synthesis of glycine and formate; the latter is necessary for the biosynthesis of deoxythymidylate (dTMP) used in DNA replication.21,22 SHMT2 has been shown to be upregulated under hypoxic conditions,23 producing glycine and CH2-THF and thereby increasing the level of synthesis of mitochondrial NADPH (through the reaction of methylenetetrahydrofolate dehydrogenase), which is necessary to counteract the increase in oxidative stress. SHMT2 may also play a crucial role in the mitochondrial formation of glycine, which is required for heme synthesis.24 SHMT2 is also expressed as a

erine hydroxymethyltransferase (SHMT, EC 2.1.2.1), whose main catalytic function is the reversible conversion of L-serine and tetrahydrofolate (THF) into glycine and 5,10methylene-tetrahydrofolate (CH2-THF),1,2 is a pivotal enzyme in one-carbon metabolism. This reaction is the main source of one-carbon groups required for the synthesis of methionine, choline, thymidylate, and purines.3 Because of its remarkable catalytic mechanism, involving pyridoxal 5′-phosphate (PLP) as a prosthetic group and THF as a cosubstrate, and its high catalytic versatility, in the past SHMT has been mainly investigated from a mechanistic and structural point of view.4−6 In recent years, research on this enzyme has focused on its role in serine/glycine one-carbon metabolism and cancer metabolic reprogramming,7 with particular attention being paid to the design of specific inhibitors to be developed as chemotherapeutic and antimalarial agents.8−15 Two genes encoding SHMT are present in humans, which express different isoforms (SHMT1 and SHMT2) sharing ∼66% amino acid sequence identity16 and apparently having very similar catalytic properties, although it is known that their © XXXX American Chemical Society

Received: October 9, 2018 Revised: November 7, 2018 Published: November 30, 2018 A

DOI: 10.1021/acs.biochem.8b01074 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry Scheme 1. Compartmentalized One-Carbon Metabolism and Role of SHMT Cytosolic and Mitochondrial Isozymesa

a

The scheme shows the reactions catalyzed by SHMT1 and SHMT1 in the context of the folate metabolism cycle taking place between the cytosol and mitochondria. The dead-end complexes responsible for the substrate inhibition observed in this work and the structures of all substrates, products, and coenzymes are also shown.

properties. However, very scarce information about SHMT2 is available.17,28 Most biochemical studies have been carried out on SHMT1, and even in this case, very little is known about how the activity of the enzyme is regulated by its substrates or other metabolites. A strong THF substrate inhibition of the Lserine to glycine reaction was first observed with Escherichia coli SHMT.29 Given the sequential random mechanism (Scheme 2) followed by the addition of substrates and release of products,30 it was attributed to the formation of an inactive enzyme−glycine−THF ternary complex. Substrate inhibition was also observed with human SHMT131 and shown to result from the rate-limiting release of glycine, which leads to formation of the enzyme−glycine−THF ternary complex. This phenomenon is strongly dependent on pH.32 It is not known whether a similar substrate inhibition takes place with L-serine

cytosolic form (SHMT2α) that lacks the mitochondrial import sequence.25 The other isoform, SHMT1, is mainly localized in the cytosol, although during the S phase of the cell cycle, it is imported together with SHMT2α in the nucleus, where it forms a complex with thymidylate synthase and dihydrofolate reductase. It supplies CH2-THF for the thymidylate cycle, participating in the synthesis of nuclear dTMP.25 Recent research showed that, in many cancer cell lines, SHMT1- and SHMT2-catalyzed reactions go in opposite directions (serine synthesis and serine degradation, respectively), thereby creating a cyclic one-carbon unit flux between the cytosol and mitochondria.26,27 Given the distinct metabolic roles played by SHMT1 and SHMT2 in different cellular compartments, it is expected that the two isozymes might show different catalytic and regulation B


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Biochemistry Scheme 2. Sequential Random Mechanism of Addition of Substrates and Release of Products Followed by SHMTa

a

All steps in the mechanism are considered to be reversible. Abbreviations: E, enzyme; G, glycine; S, L-serine; T, tetrahydrofolate; M, 5,10methylene-THF; EST and EGM, productive complexes; EGT and ESM, unproductive complexes.

reaction was measured by means of a spectrophotometric coupled assay, in which the 5,10-CH2-THF produced by the SHMT reaction was oxidized by the NADP+-dependent E. coli 5,10-methylenetetrahydrofolate dehydrogenase.36,37 Initially, all saturation curves obtained by varying one substrate at different fixed concentrations of the other substrate were independently fitted to a modified Michaelis−Menten equation (eq 1) that accounts for substrate inhibition, to acquire information about the dependence of kinetic parameters on THF and L-serine concentrations.

and the substrates involved in the reverse glycine to L-serine reaction (i.e., glycine and CH2-THF). It is also unknown whether SHMT2 shows substrate inhibition and if this depends on pH. Here we present a comparative full kinetic characterization of the forward (L-serine to glycine) and backward (glycine to Lserine) reactions catalyzed by SHMT1 and SHMT2. Our analyses yield novel information about the substrate inhibition properties of the isozymes, which might be related to their different cellular localization and regulatory mechanisms.

■

MATERIALS AND METHODS Materials. Tetrahydrofolate and 5,10-methylene tetrahydrofolate (monoglutamylated forms) were kindly provided by Merck & Cie (Schaffhausen, Switzerland). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO). Enzyme Purification. Human SHMT1 (UniProt entry P34896) and SHMT2 (UniProt entry P34897) were recombinantly expressed using E. coli as a host and purified as previously described.17,33 The coding sequence used to express human mitochondrial SHMT2 corresponds to that of isoform 3 (NCBI reference sequence NP_001159831.1), whose amino acid sequence is identical to that of the fulllength precursor (isoform 1) but lacks of the N-terminal mitochondrial import signal. The extinction coefficients at 280 nm used to determine enzyme subunit concentrations were 47565 and 43887 cm−1 M−1 for SHMT1 and SHMT2, respectively.17,34 Recombinant L-serine dehydrogenase from Pseudomonas aeruginosa was expressed as a His-tagged protein and purified by affinity chromatography in 50 mM potassium phosphate buffer (pH 8) containing 0.5 M NaCl.35 Enzyme Activity Assays. Initial Velocity Measurements of the SHMT Forward Reaction (L-serine + THF → glycine + CH2-THF). Activity assays were performed at 30 °C in 20 mM potassium phosphate buffer at the indicated pH, except in the experiments shown in Figure 1, which were performed in a mixture of 50 mM MES, 50 mM HEPES, and 50 mM CHES, brought to the indicated pH values with potassium hydroxide. All experiments were performed in at least triplicate using a Hewlett-Packard 8453 diode-array spectrophotometer (Agilent Technologies, Santa Clara, CA). The initial velocity of the

vi = Vmax

[Sv ] i K + [Sv ]jjj1 + app m k

y [Sv ] z z Ki z

app

{

(1)

where vi stands for the initial velocity, Vmax is the maximum velocity, [Sv] is the concentration of the variable substrate, appKm is the apparent Michaelis−Menten constant for the variable substrate, and appKi is the apparent substrate inhibition constant. Following this preliminary analysis, the set of curves obtained with a variable THF concentration and a fixed Lserine concentration was globally fitted to a modified Michaelis−Menten equation (eq 2) that accounts for the variation of Vmax and the THF substrate inhibition constant ) as a function of L-serine concentration (KTHF i [Ser] [THF] ji zyz z vi = Vmax jjj j αK Ser + [Ser] zz THF i k m {K + [THF]jjjj1 + m k

y [THF] z zz K iTHF z

app

{ (2)

αKSer m

where is the Km for L-serine at a saturating THF is the Michaelis constant for THF, and concentration, KTHF m THF K is the apparent THF substrate inhibition constant. app i On the other hand, the set of curves obtained with variable concentrations and a fixed THF concentration were globally fitted to eq 3

L-serine

C


Biochemistry ÄÅ ÅÅ ÅÅ ÅÅ [THF] vi = VmaxÅÅÅ ÅÅ ÅÅ αK mTHF + [THF] 1 + ÅÅÇ

(

[THF] K iTHF

)

ÑÉÑ ÑÑ ÑÑ ÑÑ ÑÑ ÑÑ ÑÑ ÑÑÖ

Article

change at 502 nm (maximum absorbance of the human SHMT−glycine−5-CHO-THF ternary complex) was measured. Measurement of the Quinonoid Intermediate Formed upon Addition of Glycine and 5,10-Methylene-THF to SHMT. The absorbance change at 493 nm (ΔAbs493 in Figures 4−6; 493 nm corresponds to the maximum absorbance wavelength of the quinonoid intermediate formed from the human SHMT−glycine−CH2-THF ternary complex) was measured at steady state upon addition of CH2-THF to samples containing either SHMT1 or SHMT2 (5 μM) and glycine. Assays, conducted in triplicate in 20 mM potassium phosphate buffer at pH 7.2 with SHMT1 and at pH 7.8 with SHMT2, were performed at 30 °C using a Multiscan GO microplate spectrophotometer (ThermoFisher Scientific, Waltham, MA). Saturation curves were fitted to a quadratic equation (eq 6) ÄÅ Å Abs493 = ΔAbs ÅÅÅ[Sv ] + [E] + Kdapp ÅÇ ÉÑ Ñ − (Kdapp + [Sv ] + [E])2 − 4[E][Sv ] ÑÑÑ ÑÖ

[Ser]

× app

(

K mSer + [Ser] 1 +

[Ser] K iSer

)

(3)

where αKTHF is the Km for THF at a saturating L-serine m concentration, KTHF is the THF substrate inhibition constant, i Ser K is the apparent Km for L-serine, and KSer i is the L-serine m app substrate inhibition constant. Initial Velocity Measurements of the SHMT Reverse Reaction (glycine + CH2-THF → L-serine + THF). A novel, direct coupled spectrophotometric assay was devised in which the NAD+ -dependent L -serine dehydrogenase from P. aeruginosa35 was used to oxidize L-serine produced by SHMT into 2-aminomethylmalonate semialdehyde. These experiments were performed in 50 mM Tris-HCl at pH 8.8, which is the optimum pH of L-serine dehydrogenase, used in the assay at a final concentration of 2 μM in the presence of 5 mM NAD+. The saturation curves obtained by varying the glycine concentration at different fixed CH2-THF concentrations were globally fitted to a modified Michaelis−Menten equation (eq 4), which is analogous to eq 2 and accounts for variation in Vmax as a function of CH2-THF concentration and for glycine substrate inhibition ij yz [CH ‐THF] zz vi = Vmax jjj CH ‐THF 2 zz j αK 2 CH THF + [ ‐ ] m 2 k { [Gly] × i [Gly] y zz K mGly + [Gly]jjjj1 + z K iGly z app k {

{

( )}

/ 2[E]

where [Sv] is the concentration of the variable substrate, [E] is the total enzyme concentration, ΔAbs is the maximum absorbance change (whose dependence on the fixed substrate concentration was analyzed with eq 7), and Kdapp is the apparent dissociation constant for the variable substrate (whose dependence on the fixed substrate concentration was analyzed with eq 8). ΔAbs = ΔAbsmax

[Sf ] [Sf ] + Kd

ij [Sf ] yzz Kdapp = (Kd − αKd)jjj1 − z + αK d j [Sf ] + K zz{ k

(4)

2‑THF where αKCH is the Km for CH2-THF at a saturating glycine m Gly concentration, KGly is the m is the Km for glycine, and appKi apparent glycine substrate inhibition constant. The set of curves obtained with variable CH2-THF concentrations and a fixed glycine concentration was globally fitted to eq 5, which accounts for variation of Vmax as a function of glycine concentration

ij yz [Gly] [CH 2‐THF] zz vi = Vmax jjjj Gly zz CH2 ‐ THF + [CH 2‐THF] k αK m + [Gly] { K m

(6)

(7)

(8)

In eqs 7 and 8, [Sf] is the concentration of the fixed substrate. Kd and αKd are the dissociation constants of the variable substrate at zero and saturating concentrations of the fixed substrate, respectively. Product inhibition experiments in the presence of different L-serine concentrations were performed by either varying the concentration of glycine (0.3−10 mM) while keeping CH2THF at a fixed concentration (0.15 mM) or varying the concentration of CH2-THF (0.05−0.15 mM) while keeping glycine at a fixed concentration (15 mM).

(5)

■

αKGly m

where is the Km for glycine at a saturating CH2-THF 2‑THF concentration and KCH is the Km for CH2-THF. m Measurements of the Quinonoid Intermediate Developed upon Addition of Glycine and 5-Formyl-THF to SHMT. This assay is based on the spectrophotometric measurement of the quinonoid intermediate that develops when glycine and a folate bind to SHMT. This intermediate, which is derived from deprotonation of glycine, yields an intense absorption band with a maximum at around 500 nm and accumulates to a measurable extent only when a folate ligand is added to SHMT, forming a ternary complex.38,39 The absorbance at 500 nm is proportional to the fraction of enzyme present as a ternary complex. When the dependence of SHMT activity on pH was assayed, 5-CHO-THF (80 μM) was added as the last component to a SHMT (5 μM) sample containing 10 mM glycine, and after a rapid manual mixing, the absorbance

RESULTS Dependence of SHMT Activity on pH. It is known that the mitochondrial matrix has an alkaline pH (pH 7.7−7.940,41) compared to that of the cytosol (pH 7.0−7.441,42). To check whether SHMT1 and SHMT2 are differently dependent on pH, two different reactions catalyzed by the isozymes were measured as a function of pH: (i) the conversion of L-serine and THF into glycine and CH2-THF (SHMT forward reaction) and (ii) the formation of the quinonoid intermediate that accumulates when both glycine and 5-formyl-THF bind to the enzyme.38,43 The results showed that, for both reactions, the pH optimum of SHMT2 is 0.5 unit higher with respect to that of SHMT1 (Figure 1A). As shown previously,32 it can be assumed that under these conditions the amino acid concentration used in the activity assays (10 mM glycine or D


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serine concentration was kept fixed and saturating (10 mM). Measurements were taken at pH 6.9, 7.2, 7.8, and 8.4 (Figure 1B,C). At pH 7.2, with both isozymes a strong THF substrate inhibition was observed. At 400 μM THF, the activity of the enzymes was half of the maximum activity measured. However, a remarkable difference was observed between SHMT1 and SHMT2 in the pH dependence of THF substrate inhibition. In particular, while SHMT1, as previously reported,32 shows a marked decrease in the level of substrate inhibition at high pH values, inhibition of SHMT2 persists even at pH 8.4. Steady-State Kinetic Analysis of the Serine Hydroxymethyltransferase Forward Reaction (L-serine + THF → glycine + CH2-THF). The initial velocity of the SHMT forward reaction was measured with either SHMT1 (at pH 7.2) or SHMT2 (at pH 7.8, which is the pH value in the mitochondrial matrix, and also at pH 7.2, to allow a direct comparison with SHMT1), varying one substrate while keeping the other fixed at different concentrations, so that two different sets of saturation curves were obtained. When using SHMT1 as a catalyst, a clear substrate inhibition was observed in the set of curves obtained with variable THF concentrations, which became more marked as the fixed Lserine concentration was increased (Figure 2A). Initially, each curve of this set was independently fitted to a modified Michaelis−Menten equation that accounts for uncompetitive inhibition (eq 1), yielding estimates of the apparent Vmax, Km for THF, and Ki for THF substrate inhibition. As expected from a bireactant system, Vmax showed an increasing hyperbolic dependence on L-serine concentration (data not shown), while the value of Ki showed a decreasing hyperbolic dependence on L-serine concentration. Because of the known synergistic binding of substrates to the enzyme,30 the value of Km for THF was expected to decrease hyperbolically as the L-serine concentration was increased; however, our analysis failed to highlight such a correlation, probably because of the large number of variables in the fitting procedure and the experimental noise present in the data. On the basis ogf this information, a modified Michaelis−Menten equation was derived and used to globally fit the data (eq 2). In the leastsquares minimization procedure, Vmax, αKSer m (the α prefix represents the factor by which the Km for this substrate is decreased by a saturating concentration of the other substrate), and KmTHF were shared parameters. Given the previous was assumed not to vary as a function of observations, KTHF m L-serine concentration and taken as an intermediate value between the Km for THF at zero and saturating L-serine concentrations (αKmTHF). The apparent THF substrate inhibition constant (appKTHF in eq 2) was not a shared i parameter and allowed to vary in the fitting as a function of Lvalues produced by the serine concentration. The appKTHF i fitting procedure, which showed a decreasing hyperbolic dependence on L-serine concentration, were in their turn fitted to a hyperbolic equation (Figure 2A, inset) to obtain an estimate of Ki at an infinite L-serine concentration, which . Table 1 reports the values corresponds to the authentic KTHF i of all parameters produced by the fitting procedure. The second set of saturating curves, obtained with variable Lserine concentrations and a fixed THF concentration, showed a completely different pattern (Figure 2B). In this case, substrate inhibition by L-serine becomes evident as the THF concentration is decreased and is particularly marked at the lowest THF concentration (0.012 mM). Fitting of each single curve to eq 1 revealed that the apparent Vmax increases as the

Figure 1. Dependence of SHMT1 and SHMT2 activity on pH. (A) The SHMT forward reaction (L-serine + THF → glycine + CH2THF) was measured with 0.5 μM enzyme, 10 mM L-serine, and 65 μM THF (black symbols; SHMT1, filled circles; SHMT2, empty circles). The formation of the quinonoid intermediate was measured using 5 μM enzyme, 10 mM glycine, and 80 μM 5-CHO-THF (red symbols; SHMT1, filled circles; SHMT2, empty circles). Both reactions were performed at 30 °C. Activity values, reported as a percentage of the maximum activity measured, are the average ± the standard deviation of three independent measurements. (B and C) The initial velocity of the SHMT forward reaction was measured at the indicated pH values with either SHMT1 or SHMT2 (0.2 μM) using a fixed, saturating L-serine concentration (10 mM) and a varied THF concentration. Also in this case, as in all other figures, experimental values are the average ± the standard deviation of three independent measurements. The solid lines are nonlinear leastsquares fits to the experimental data obtained according to eq 1. L-serine)

was saturating at all pH values; therefore, the dependence of activity on pH may be mainly attributed to changes in folate substrate affinity and/or reaction turnover. In parallel, to examine the pH dependence of THF substrate inhibition, the initial velocity of the SHMT forward reaction was measured at different THF concentrations while the LE


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Figure 2. Steady-state kinetic measurements of the SHMT forward reaction (L-serine + THF → glycine + CH2-THF) catalyzed by SHMT1 (A−C) and SHMT2 (D−F) (0.2 μM) at 30 °C in 20 mM KPi buffer at pH 7.2 and 7.8, respectively. (A) Saturation curves obtained with SHMT1 by varying the THF concentration while keeping L-serine fixed at different concentrations [10 (○), 5 (▽), 2.5 (◇), 0.625 (□), 0.312 (△), 0.156 (∗), and 0.078 (●) mM]. The solid lines through the experimental points were obtained by global fitting of data to eq 2, as explained in the text. The ) resulting from the fitting procedure, which were analyzed inset shows the values of the apparent THF substrate inhibition constant (appKTHF i according to the equation of a decreasing hyperbolic curve (solid line). (B) Saturation curves obtained with SHMT1 by varying the L-serine concentration while keeping THF fixed at different concentrations [0.485 (○), 0.242 (▽), 0.194 (◇), 0.121 (□), 0.097 (△), 0.036 (●), 0.024 (■), and 0.012 (▲) mM]. The solid curves were obtained by global fitting of data to eq 4, as explained in the text. (C) Dependence of the apparent Km for L-serine and the apparent Ki for L-serine substrate inhibition (inset) on THF concentration. (D) Saturation curves obtained with SHMT2 by varying the THF concentration while keeping L-serine fixed at different concentrations [5 (▽), 2.5 (◇), 0.625 (□), 0.312 (△), and 0.156 (∗) . (E) Saturation curves obtained with mM]. As explained for SHMT1, data were globally fitted to eq 2 and the inset shows the values of appKTHF i SHMT2 by varying the L-serine concentration while keeping THF fixed at different concentrations [0.380 (○), 0.190 (▽), 0.095 (◇), 0.048 (□), 0.024 (△), and 0.012 (●) mM]. The solid curves were obtained by global fitting of data to eq 3. (F) Dependence of the apparent Km for L-serine and the apparent Ki for L-serine (inset) on THF concentration.

inhibition at a saturating L-serine concentration) were shared parameters in the least-squares minimization procedure. On the other hand, the apparent Km for L-serine (appKSer m ) and the apparent L-serine substrate inhibition constant (appKSer i ) were allowed to vary in the fitting. The value of appKSer m returned by the fitting shows a decreasing hyperbolic dependence on THF concentration (Figure 2C), starting from a maximum value (KmSer) at a zero THF concentration and asymptotically approaching a minimum value at a saturating THF concentration (αKSer m ). These values were extrapolated and shows an are reported in Table 1. The value of appKSer i

THF concentration is increased, reaching a maximum value at ∼0.097 mM THF, but then decreases as the THF concentration is further increased (data not shown); the THF substrate inhibition is clearly responsible for this behavior. As expected, because of the synergistic binding of substrates, the apparent Km for L-serine decreases hyperbolically as a function of THF concentration. Given these observations, data were globally fitted to a modified Michaelis−Menten equation that accounts for the variation in the apparent Vmax and for L-serine substrate inhibition (eq THF (Ki for THF substrate 3). In the fitting, Vmax, αKTHF m , and Ki F


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to acquire information about the dependence of kinetic parameters on substrate concentration. With variable glycine concentrations, an increasing hyperbolic dependence of Vmax on substrate concentration was observed (data not shown). Glycine substrate inhibition is visible at the lowest CH2-THF concentrations (panels A and D of Figure 3 for SHMT1 and SHMT2, respectively). No clear dependence of Km for glycine on CH2-THF concentration was observed (data not shown). Data were globally fitted to eq 4. The apparent glycine substrate inhibition constant (appKGly i ) showed an exponential dependence on CH2-THF concentration (Figure 3B,E). With variable CH2-THF concentrations, the same hyperbolic dependence of Vmax on the fixed substrate (glycine in this case) was observed, but no substrate inhibition was visible in the CH2-THF concentration range used in the assay (≤0.65 mM), which was limited by the high absorbance of this folate substrate that interfered with the spectrophotometric measurements. No apparent dependence of the Km for CH2-THF on glycine concentration was observed. Data were globally fitted to eq 5 (Figure 3C,F). Kinetic parameters obtained from global fitting of both sets of data are listed in Table 1. Binding of L-Serine to the Enzyme−CH2-THF Complex. The L-serine substrate inhibition observed in the forward serine hydroxymethyltransferase reaction (L-serine + THF → glycine + CH2-THF) with both SHMT1 and SHMT2 suggests that an abortive enzyme−serine−CH2-THF ternary complex may be formed (ESM in Scheme 2). The formation of this complex had been previously suggested with rabbit SHMT1 on the basis of product inhibition experiments performed at pH 8.3.30 To verify and extend this observation to both human SHMT isoforms at pH values close to those present in the cytosol and in the mitochondrial matrix, L-serine product inhibition experiments for the reverse SHMT reaction were performed using the following kinetic assay. It is known that, when glycine binds to SHMT, a quinonoid intermediate derived from deprotonation of the α-carbon is formed that maximally absorbs between 490 and 505 nm. The concentration of this intermediate is greatly increased if THF, 5formyl-THF, or 5-methyl-THF is added to the enzyme, forming an abortive enzyme−glycine−folate ternary complex that cannot develop into products.38,44 We noticed that CH2THF has the same effect of increasing the concentration of the quinonoid intermediate when added to solutions of either SHMT1 or SHMT2 in the presence of glycine. However, in this case, the formed ternary complex is a productive reaction intermediate in the conversion of glycine and CH2-THF into Lserine and THF, and the quinonoid intermediate remains at a constant concentration during the steady state (data not shown). We performed a series of experiments (at pH 7.2 with SHMT1 and at pH 7.8 with SHMT2) in which the absorbance change at 493 nm (ΔAbs493) was measured upon mixing of the enzyme with glycine and CH2-THF at different concentrations (ΔAbs493 is the difference between the absorbance at 493 nm measured before addition of CH2-THF and after reaching the steady state of the reaction). Two families of saturation curves were obtained by varying the concentration of one substrate while keeping the other fixed (Figure 4A,C for SHMT1; Figure 5A,C for SHMT2). Each curve was independently analyzed with a quadratic equation (eq 6), obtaining values of the apparent Kd of the variable substrate and the maximum absorbance change as a function of the fixed substrate concentration. These parameters, which show a hyperbolic

Table 1. Steady-State Kinetic Parameters of the Forward and Reverse Hydroxymethyltransferase Reactions Catalyzed by SHMT1 and SHMT2 L-Ser

+ THF → Gly + CH2-THF (forward reaction)

value for SHMT1 (min−1 or mM) kcata KTHF m αKTHF m a KSer m Ser αKm a KTHF i kcat/KTHF m kcat/ THF αKm kcat/KSer m kcat/αKSer m

value for SHMT2 (min−1 or mM)

pH 7.2

pH 7.2

pH 7.8

374 ± 20 0.033 ± 0.003 0.007 ± 0.003 4.8 ± 2.5 0.131 ± 0.008 0.258 ± 0.012 11330 53430

850 ± 0.1 0.223 ± 0.085 0.053 ± 0.017 2.2 ± 0.2 0.396 ± 0.080 0.034 ± 0.002 3810 16040

813 ± 0.2 0.032 ± 0.005 0.020 ± 0.008 0.45 ± 0.05 0.158 ± 0.05 0.099 ± 0.017 25400 40650

80 390 1810 2850 2150 5150 Gly + CH2-THF → L-Ser + THF (backward reaction) value for SHMT1 (min−1 or mM)

a kGly cat CH2‑THF Km 2‑THF αKCH m Gly Km αKGly m CH ‑THF kcat/Km 2 kcat/ 2‑HF αKCH m kcat/KGly m kcat/αKGly m

value for SHMT2 (min−1 or mM)

pH 8.8

pH 8.8

11.3 ± 0.3 0.291 ± 0.018 0.159 ± 0.014 0.556 ± 0.033 0.514 ± 0.028 39 71

30.07 ± 2.31 0.98 ± 0.08 0.98 ± 0.13 0.66 ± 0.04 0.47 ± 0.02 31 31

20 22

46 64

Average of the two values determined by the fitting of data sets obtained by varying one substrate while keeping the other fixed. a

increasing exponential dependence on THF concentration (Figure 2C, inset). Analogous results were obtained with SHMT2 at pH 7.2 and 7.8, although with this isozyme a more pronounced THF substrate inhibition and a higher kcat were observed in comparison to those of SHMT1 at both pH values. All saturation curves were analyzed according to the procedures previously described for SHMT1 (Figure 2D−F and Table 1). It is clear that the catalytic efficiency of SHMT2 with both Lserine and THF is much higher at pH 7.8 than at pH 7.2. Conversely, it is known that SHMT1 shows the opposite behavior.32 Steady-State Kinetic Analysis of the Serine Hydroxymethyltransferase Backward Reaction (glycine + CH2THF → L-serine + THF). We also analyzed the SHMT backward reaction in which the enzyme catalyzes the conversion of 5,10-methylene-THF and glycine to THF and L-serine. To perform the kinetic measurements, we set up a novel coupled assay in which the L-serine product is oxidized to 2-aminomethylmalonate semialdehyde by the NAD+dependent L-serine dehydrogenase from P. aeruginosa. Because of the alkaline pH optimum of L-serine dehydrogenase, this coupled assay was performed at pH 8.8 (see Materials and Methods for details). Also in this case, two sets of saturation curves were obtained, depending on whether glycine or CH2THF was the variable substrate in the assay (Figure 3). Initially, all saturation curves were analyzed according to eq 1 G


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Figure 3. Steady-state kinetic measurements of the SHMT backward reaction (glycine + CH2-THF → L-serine + THF) catalyzed by SHMT1 (A− C) and SHMT2 (D−F) (2 μM). Kinetic measurements were taken at pH 8.8 in 50 mM Tris HCl buffer at 30 °C. (A) With SHMT1, the glycine concentration was varied while keeping CH2-THF fixed at different concentrations [0.490 (○), 0.326 (▽), 0.163 (◇), 0.114 (□), 0.081 (△), and 0.049 (∗) mM]. The solid lines through the experimental points were obtained by global fitting of data to eq 4, as explained in the text. (B) Values of the apparent glycine substrate inhibition constant (appKGly i ) resulting from the fitting procedure, which show an exponential dependence on CH2THF concentration. (C) Saturation curves obtained with SHMT1 by varying the CH2-THF concentration while keeping glycine fixed at different concentrations [20 (○), 5 (▽), 2.5 (◇), 1.25 (□), 0.63 (△), 0.31 (∗), and 0.16 (●) mM]. The solid curves were obtained by global fitting of data to eq 5, as explained in the text. (D) With SHMT2, the glycine concentration was varied while keeping CH2-THF fixed at different concentrations [0.490 (○), 0.326 (▽), 0.163 (◇), 0.114 (□), 0.081 (△), and 0.049 (∗) mM]. The solid lines through the experimental points were obtained by global fitting of data to eq 4. (E) Values of the apparent glycine substrate inhibition constant (appKGly i ) resulting from the fitting procedure. (F) Saturation curves obtained with SHMT2 by varying the CH2-THF concentration while keeping glycine fixed at different concentrations [20 (○), 5 (▽), 2.5 (◇), 1.25 (□), 0.63 (△), 0.31 (∗), and 0.16 (●) mM]. Solid curves were obtained by global fitting of data to eq 5, as explained in the text.

dependence on the fixed substrate concentration (Figure 4B,D for SHMT1; Figure 5B,D for SHMT2), were analyzed using eqs 7 and 8 to obtain Kd and αKd for both ligands (Table 2). We observed that the addition of L-serine to a reaction mixture containing glycine and CH2-THF decreases the concentration of the quinonoid intermediate. We took advantage of this observation to perform a series of product inhibition experiments. Measurements of absorbance at 493 nm were taken at different L-serine concentrations, varying the concentration of one substrate while keeping the other substrate at a fixed, saturating concentration. Double-reciprocal

plots of absorbance changes obtained as a function of the varying ligand concentration at different L-serine concentrations clearly show, with both SHMT1 and SHMT2, that Lserine competes with glycine (Figure 6A) but not with CH2THF (Figure 6B). Secondary plots of slopes (Figure 6C,D) and intercepts (Figure 6E) calculated from double-reciprocal plots gave estimates of inhibition constants related to binding of L-serine to the free enzyme (Ki) and to the enzyme−CH2THF complex (Kii) (Table 2). The same set of experiments performed with SHMT2 in 20 mM KPi buffer at pH 7.8 gave an identical inhibition pattern H


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Biochemistry

Figure 4. Determination of glycine and CH2-THF binding parameters for the SHMT backward reaction catalyzed by SHMT1. The absorbance change at 493 nm measured upon addition of CH2-THF to samples containing SHMT (5 μM) and glycine at the steady state of the SHMT backward reaction. Assays were performed in triplicate at 30 °C in 20 mM KPi buffer at pH 7.2. (A) Measurements were taken at varying CH2THF concentrations while keeping glycine at different fixed concentrations [10 (○), 5 (▽), 1.25 (◇), 0.625 (□), and 0.312 (△) mM]. The obtained saturation curves were fitted to eq 6, producing estimates of the maximum absorbance change at 493 nm (ΔAbsmax) and the apparent 2‑THF ) as a function of glycine concentration. (B) Parameters and solid lines that result from their leastdissociation constant of CH2-THF (KCH dapp squares fitting to eqs 7 and 8, respectively, which yielded Kd and αKd for glycine. (C) Measurements were taken at varying glycine concentrations while keeping CH2-THF at different fixed concentrations [0.133 (○), 0.107 (▽), 0.080 (◇), 0.053 (□), 0.032 (△), 0.021 (∗), 0.011 (●), and 0.005 (■) mM]. Data were fitted to eq 6, producing the values of ΔAbsmax and KGly dapp as a function of CH2-THF concentration shown in panel D. These were fitted to eqs 7 and 8, respectively, yielding Kd and αKd for CH2-THF.

μM THF, the pH optimum is 8.5, 0.5 pH unit higher that the pH optimum of SHMT1 (Figure 1A). The pH−activity profiles obtained by measuring quinonoid formation upon addition of glycine and 5-CHO-THF are simpler to interpret, because no substrate inhibition can be present with these ligands that are not transformed by SHMT but establish a binding equilibrium with the enzyme. Also in this case, the pH optimum of SHMT1 (∼7.5) is 0.5 pH unit lower than that of SHMT2 (∼8.0). Given such considerations, it may be hypothesized that these different properties of SHMT1 and SHMT2 represent an adaptation of the two isoforms to their respective cellular environments. THF substrate inhibition is due to the formation of an enzyme−glycine−THF dead-end complex (EGT in Scheme 2). The fact that this inhibition is completely absent in the SHMT from Plasmodium vivax32 suggests that THF substrate inhibition is not just an inevitable consequence of the sequential random mechanism obeyed by SHMT45 but that it may represent a form of enzyme regulation in particular organisms and cellular compartments. It is known that substrate inhibition, as well as product inhibition, often has important biological functions.46−48 In particular, in folate metabolism, substrate inhibition is generally believed to represent a mechanism of storage of folate units. When the cellular total folate concentration decreases, folate is released from the enzymes, which are consequently reactivated, maintaining the velocities in the folate cycle, despite the loss of total folate.46 In this view, the conservation of THF

(data not shown), which allowed the estimation of the inhibition parameters listed in Table 2.

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DISCUSSION Our comparative characterization of SHMT1 and SHMT2 highlighted several interesting differences between the two SHMT isoforms. Although both isozymes are strongly affected by THF substrate inhibition when catalyzing the SHMT forward reaction, measurements of enzyme activity as a function of pH show a clearly different behavior of SHMT1 and SHMT2 (Figure 1). While with SHMT1, as previously reported,32 increasing pH values strongly attenuate THF substrate inhibition, with SHMT2, pH changes do not affect substrate inhibition to the same extent. Interestingly, such behavior influences the pH−activity profiles of SHMT1 and SHMT2, which differently depend on THF concentration. As shown in Figure 1B, in the case of SHMT1, below 20 μM THF, the enzyme activity decreases as the pH is increased from 6.9 to 8.4. Between 20 and 100 μM THF, the pH optimum varies as a function of THF concentration. Under the experimental conditions used to generate the data shown in Figure 1A (65 μM THF), the pH optimum is around 8.0. Above 100 μM THF, the pH profile is reversed and the activity increases as the pH is increased from 6.9 to 8.4. With SHMT2, the activity changes as a function of pH are much more limited. At THF concentrations of 20 μM THF, the activity increases as the pH is increased, so that at 65 I


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Biochemistry

Figure 5. Determination of glycine and CH2-THF binding parameters for the SHMT backward reaction catalyzed by SHMT2. Experimental conditions were as described in the legend of Figure 6. Assays were performed in triplicate at 30 °C in 20 mM KPi buffer at pH 7.8. (A) Measurements were taken at varying CH2-THF concentrations while keeping glycine at different fixed concentrations [5 (▽), 2.5 (○), 1.25 (◇), 0.62 (□), 0.3 (△), and 0.16 (∗) mM]. The obtained saturation curves were fitted to eq 6, yielding estimates of the maximum absorbance change at 2‑THF ) as a function of glycine concentration. (B) Parameters and solid 493 nm (ΔAbsmax) and the apparent dissociation constant of CH2-THF (KCH dapp lines that result from their least-squares fitting to eqs 7 and 8, respectively, which yielded Kd and αKd for glycine. (C) Measurements were taken at varying glycine concentrations while keeping CH2-THF at different fixed concentrations [0.1 (○), 0.05 (▽), 0.025 (◇), 0.012 (□), 0.006 (△), and 0.003 (∗) mM]. Data were fitted to eq 6, yielding values of ΔAbsmax and KGly dapp as a function of CH2-THF concentration shown in panel D. These were fitted to eqs 7 and 8, respectively, yielding Kd and αKd for CH2-THF.

residues 271−287,32 because this is present in human SHMT1 but absent in Pl. vivax SHMT. However, this flap motif is also absent in E. coli SHMT, which shows THF substrate inhibition.29 We believe that the presence of this flap motif is related to the quaternary structure of SHMT, which is homotetrameric in human, rabbit, and mouse SHMTs, all presenting the flap motif,2,49,50 but is homodimeric in E. coli and Pl. vivax SHMTs. Therefore, the structural basis of substrate inhibition in SHMT remains unclear. Our steady-state kinetic analyses of SHMT forward and backward reactions were performed using equations that take into account substrate inhibition and synergistic binding of substrates. This allowed the global fitting of data, taking into consideration the entire extent of the saturation curves (without cutting out the part in which substrate inhibition is predominant), and therefore the determination of all kinetic parameters (Table 1). Our studies extended the previous analyses performed on rabbit SHMT145 and human SHMT132 and yielded new information about SHMT2. With both isozymes, we detected a previously unobserved L-serine substrate inhibition (in the L-serine + THF → glycine + CH2-THF direction) and glycine substrate inhibition (in the glycine + CH2-THF → L-serine + THF direction). The Lserine substrate inhibition may be attributed to the formation of an enzyme−serine−CH2-THF dead-end complex (ESM in Scheme 2). Such a possibility had been previously proposed by other authors,45 implying that the SHMT active site can simultaneously accommodate the one-carbon groups present

Table 2. Binding and Inhibition Constants Concerning Substrates of the SHMT Backward Reaction (glycine and CH2-THF) and L-Serine Product Inhibitiona value for SHMT1 (mM) kGly d b αKGly d 2‑THF KCH d 2‑THFb αKCH d Serc Ki KSer ii

value for SHMT2 (mM)

pH 7.2

pH 7.8

1.77 ± 0.11 0.71 ± 0.12 0.084 ± 0.048 0.0082 ± 0.0025 0.368 ± 0.091 4.1 ± 0.15

3.53 ± 0.46 0.78 ± 0.08 0.202 ± 0.043 0.0080 ± 0.0005 0.602 ± 0.37 4.44 ± 0.15

a

Data were determined by measuring the quinonoid intermediate formed in the steady state of the reaction, as detailed in the text. b Average of the two values yielded by the fitting of data sets obtained by varying one substrate while keeping the other fixed. cAverage value obtained from the inhibition constant extrapolated from both sets of data obtained by varying one ligand while keeping the other fixed.

substrate inhibition by SHMT2 at alkaline pH may represent a form of adaptation of this isozyme to the mitochondrial matrix, whose pH is 7.7−7.9, which allows SHMT to contribute to folate metabolism regulation also in this environment (Scheme 1). It should be noticed here that 5-methyl-THF and 5-CHOTHF bind to the SHMT−glycine complex and may be important physiological SHMT inhibitors.45 It has been proposed that THF substrate inhibition in SHMT1 may be linked to a particular flap motif made of J


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Biochemistry

Figure 6. L-Serine product inhibition experiments for the SHMT backward reaction catalyzed by SHMT1. Experiments were performed with both SHMT1 and SHMT2 as explained in Figure 5, except that L-serine was also included in the assays. Only experiments performed with SHMT1 are shown in the figure. (A) Double-reciprocal plot of data obtained by varying the concentration of glycine (from 0.31 to 10 mM) while keeping the CH2-THF concentration fixed (0.15 mM) in the presence of different L-serine concentrations [20 (▲), 10 (■), 5 (●), 2.5 (∗), 1.25 (▽), 0.6 (◇), 0.31 (□), 0.16 (△), and 0 (○) mM]. (B) Double-reciprocal plot of data obtained by varying the CH2-THF concentration (from 0.05 to 0.15 mM) while keeping the glycine concentration fixed (15 mM) in the presence of different L-serine concentrations [20 (▲), 10 (■), 5 (●), 2.5 (∗), 1.25 (▽), 0.6 (□), and 0 (○) mM]. (C and D) Secondary plots of slopes as a function of L-serine concentration obtained from fitting of data shown in panels A and B, respectively. In both plots, the intercept on the X-axis gives an estimate of the inhibition constant related to binding of L-serine to the free enzyme (Ki), whose average is shown in Table 2. (E) Secondary plot of intercepts obtained from fitting of data shown in panel B as a function of L-serine concentration. The intercept on the X-axis gives an estimate of the inhibition constants related to binding of L-serine to the enzyme−CH2-THF complex (Kii).

increases and readily forms a dead-end complex with THF (EGT in Scheme 2). In the case of L-serine inhibition, an enzyme−serine−CH2-THF complex (ESM in Scheme 2) is formed upon binding of L-serine to the enzyme−CH2-THF complex. However, the enzyme−CH2-THF complex is present in only very small amounts, because most of the enzyme at the steady state exists as a complex with glycine, and the amount is further decreased by high THF concentrations that drag the enzyme into the enzyme−glycine−THF complex. This might be the reason why L-serine substrate inhibition is visible only at low THF concentrations. The rate-limiting step of the SHMT backward reaction is not known. Regardless, in this direction no CH2-THF substrate inhibition was observed under the conditions used for the assay (Figure 3C,F). Therefore, we speculate that L-serine release should not be the rate-liming step of the backward reaction, because the enzyme−serine complex needed for CH2-THF substrate inhibition does not accumulate. Moreover, glycine substrate inhibition was detected only at low CH2-THF concentrations (Figure 3A,D). Evidently, the release of THF is not the ratedetermining step in the SHMT backward reaction, and the enzyme−THF complex does not accumulate. Because THF and CH2-THF compete to bind to SHMT, high concentrations of CH2-THF are expected to drag the enzyme into complexes

on both the amino acid and the folate substrates. According to this hypothesis, here we demonstrate that L-serine competes with glycine in the SHMT backward reaction but does not compete with CH2-THF (Figure 6). This behavior also confirms that SHMT follows a sequential random mechanism of addition of substrates and release of products.51 Moreover, we determined the inhibition constants related to binding of Lserine to the free enzyme (Ki) and to the enzyme−CH2-THF complex (Kii) (Table 1). The higher value of Kii with respect to Ki indicates that some steric hindrance between the two onecarbon groups does take place. The glycine substrate inhibition observed in the SHMT backward reaction might be due to the formation of the same enzyme−glycine−THF dead-end complex that is responsible for THF substrate inhibition. With both L-serine and glycine, substrate inhibition is visible only at low concentrations of the folate cosubstrate, THF in the case of L-serine (Figure 2B,E) and CH2-THF in the case of glycine (Figure 3A,D). This is different from what was observed in the case of THF substrate inhibition, which becomes stronger as the concentration of the L-serine cosubstrate is increased (Figure 2A,D). The rate-limiting step in the SHMT forward reaction is the release of glycine.32 Therefore, as the L-serine concentration is increased, the fraction of enzyme present as a complex with glycine also K


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(4) Schirch, V. (1998) Mechanism of folate-requiring enzymes in onecarbon metabolism, Vol. 2, Academic Press, San Diego. (5) Florio, R., di Salvo, M. L., Vivoli, M., and Contestabile, R. (2011) Serine hydroxymethyltransferase: a model enzyme for mechanistic, structural, and evolutionary studies. Biochim. Biophys. Acta, Proteins Proteomics 1814, 1489−96. (6) Appaji Rao, N., Ambili, M., Jala, V. R., Subramanya, H. S., and Savithri, H. S. (2003) Structure-function relationship in serine hydroxymethyltransferase. Biochim. Biophys. Acta, Proteins Proteomics 1647, 24−9. (7) Amelio, I., Cutruzzola, F., Antonov, A., Agostini, M., and Melino, G. (2014) Serine and glycine metabolism in cancer. Trends Biochem. Sci. 39, 191−8. (8) Paiardini, A., Tramonti, A., Schirch, D., Guiducci, G., di Salvo, M. L., Fiascarelli, A., Giorgi, A., Maras, B., Cutruzzola, F., and Contestabile, R. (2016) Differential 3-bromopyruvate inhibition of cytosolic and mitochondrial human serine hydroxymethyltransferase isoforms, key enzymes in cancer metabolic reprogramming. Biochim. Biophys. Acta, Proteins Proteomics 1864, 1506−17. (9) Paiardini, A., Fiascarelli, A., Rinaldo, S., Daidone, F., Giardina, G., Koes, D. R., Parroni, A., Montini, G., Marani, M., Paone, A., McDermott, L. A., Contestabile, R., and Cutruzzola, F. (2015) Screening and in vitro testing of antifolate inhibitors of human cytosolic serine hydroxymethyltransferase. ChemMedChem 10, 490−7. (10) Daidone, F., Florio, R., Rinaldo, S., Contestabile, R., di Salvo, M. L., Cutruzzola, F., Bossa, F., and Paiardini, A. (2011) In silico and in vitro validation of serine hydroxymethyltransferase as a chemotherapeutic target of the antifolate drug pemetrexed. Eur. J. Med. Chem. 46, 1616−21. (11) Ducker, G. S., Ghergurovich, J. M., Mainolfi, N., Suri, V., Jeong, S. K., Hsin-Jung Li, S., Friedman, A., Manfredi, M. G., Gitai, Z., Kim, H., and Rabinowitz, J. D. (2017) Human SHMT inhibitors reveal defective glycine import as a targetable metabolic vulnerability of diffuse large B-cell lymphoma. Proc. Natl. Acad. Sci. U. S. A. 114, 11404−9. (12) Marani, M., Paone, A., Fiascarelli, A., Macone, A., Gargano, M., Rinaldo, S., Giardina, G., Pontecorvi, V., Koes, D., McDermott, L., Yang, T., Paiardini, A., Contestabile, R., and Cutruzzola, F. (2016) A pyrazolopyran derivative preferentially inhibits the activity of human cytosolic serine hydroxymethyltransferase and induces cell death in lung cancer cells. Oncotarget 7, 4570−83. (13) Witschel, M. C., Rottmann, M., Schwab, A., Leartsakulpanich, U., Chitnumsub, P., Seet, M., Tonazzi, S., Schwertz, G., Stelzer, F., Mietzner, T., McNamara, C., Thater, F., Freymond, C., Jaruwat, A., Pinthong, C., Riangrungroj, P., Oufir, M., Hamburger, M., Maser, P., Sanz-Alonso, L. M., Charman, S., Wittlin, S., Yuthavong, Y., Chaiyen, P., and Diederich, F. (2015) Inhibitors of plasmodial serine hydroxymethyltransferase (SHMT): cocrystal structures of pyrazolopyrans with potent blood- and liver-stage activities. J. Med. Chem. 58, 3117−30. (14) Ma, X., Ahmed, S., and Wohland, T. (2011) EGFR activation monitored by SW-FCCS in live cells. Front. Biosci., Elite Ed. E3, 22− 32. (15) Tramonti, A., Paiardini, A., Paone, A., Bouzidi, A., Giardina, G., Guiducci, G., Magnifico, M. C., Rinaldo, S., McDermott, L., Menendez, J. A., Contestabile, R., and Cutruzzola, F. (2018) Differential inhibitory effect of a pyrazolopyran compound on human serine hydroxymethyltransferase-amino acid complexes. Arch. Biochem. Biophys. 653, 71−9. (16) Garrow, T. A., Brenner, A. A., Whitehead, V. M., Chen, X. N., Duncan, R. G., Korenberg, J. R., and Shane, B. (1993) Cloning of human cDNAs encoding mitochondrial and cytosolic serine hydroxymethyltransferases and chromosomal localization. J. Biol. Chem. 268, 11910−6. (17) Giardina, G., Brunotti, P., Fiascarelli, A., Cicalini, A., Costa, M. G., Buckle, A. M., di Salvo, M. L., Giorgi, A., Marani, M., Paone, A., Rinaldo, S., Paiardini, A., Contestabile, R., and Cutruzzola, F. (2015) How pyridoxal 5′-phosphate differentially regulates human cytosolic

with this folate substrate and therefore decrease the concentration of the enzyme−THF complex that binds glycine and forms the enzyme−glycine−THF dead-end complex (EGT in Scheme 2). Analysis of the SHMT forward reaction shows that SHMT2 is a more efficient catalyst (in terms of kcat/Km) at pH 7.8 than at pH 7.2 (Table 1). Although kcat is very similar at these pH values, Km and αKm for both substrates are smaller at pH 7.8. It should be noticed that at pH 7.2 the catalytic efficiency of SHMT2 is smaller than that of SHMT1 at the same pH.

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CONCLUSIONS Our observations clearly indicate that SHMT2 is adapted to the mitochondrial matrix environment and that SHMT2α in the cytosol, which is structurally identical to the processed SHMT2 protein located in mitochondria, is a poorer catalyst compared to SHMT1. This raises concerns about the actual catalytic role of SHMT2α in the cytosol. The particular substrate inhibition mechanism shown by SHMT1 and SHMT2 may be relevant in the regulation of the activity of each isozyme in the compartmentalized folate metabolism of the cytosol and mitochondria.

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AUTHOR INFORMATION

Corresponding Authors

*Dipartimento di Scienze Biochimiche “A. Rossi Fanelli”, Sapienza Università di Roma, Piazzale Aldo Moro 5, 00185 Roma, Italy. E-mail: [email protected]. *E-mail: [email protected]. ORCID

Francesca Cutruzzolà: 0000-0002-4621-2135 Roberto Contestabile: 0000-0002-5235-9993 Funding

This work was supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC-IG2015 n. 16720 to F.C.) and by funds from Sapienza University of Rome (RP11715C644A5CCE to F.C.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The E. coli bacterial clone expressing recombinant L-serine dehydrogenase from P. aeruginosa was kindly provided by Prof. Alexander F. Yakunin (Department of Chemical Engineering & Applied Chemistry, University of Toronto, Toronto, ON). The authors also thank Merck & Cie for providing us with pure 5CHO-THF, 5,10-methylene-THF, and THF.

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ABBREVIATIONS PLP, pyridoxal 5′-phosphate; SHMT, serine hydroxymethyltransferase; SHMT1, cytosolic SHMT; SHMT2, mitochondrial SHMT; THF, tetrahydrofolate; CH2-THF, 5,10-methyleneTHF; 5-CHO-THF, 5-formyl-THF.

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REFERENCES

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Human Cytosolic and Mitochondrial Serine Hydroxymethyltransferase

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