Purification,physicochemical and regulatory properties of serine hydroxymethyltransferase from sheep liver

Serine hydroxymethyltransferase (EC 2.1.2.1) was purified from the cytosolic fraction of sheep liver by ammonium sulphate fractionation, CM-Sephadex chromatography, gel filtration using Ultrogel ACA 34 and Blue Sepharose affinity chromatography. The homogeneity of the enzyme was rigorously established by Polyacrylamide gel and sodium dodecyl sulphate-polyacrylamide gel electrophoresis, isoelectrofocusing, ultracentrifugation, immunodiffusion and Immunoelectrophoresis. The enzyme was a homotetramer with a molecular weight of 210,000 ±5000. The enzyme showed homotropic cooperative interactions with tetrahydrofolate (n_H =2.8) and a hyperbolic saturation pattern with L-serine. At the lowest concentration of tetrahydrofolate used (0.2 mM), only 5% of the added folate was oxidized during preincubation and assay. TheⁿH value was independent of the time of preincubation. Preincubation of the enzyme with serine resulted in a partial loss of the cooperative interactions (n_H =1.6) with tetrahydrofolate. The enzyme was regulated allosterically by interaction with nicotinamide nucleotides; NADH was a positive effector while NAD+ was a negative allosteric effector. The subunit interactions were retained even at the temperature optimum of 60‡C unlike in the case of the monkey liver enzyme, where these interactions were absent at higher temperatures. D-Cycloserine, a structural analogue of serine caused a sigmoid pattern of inhibition, in contrast with the observations on the monkey liver enzyme. Cibacron blue F3GA completely inhibited the enzyme and this inhibition could be reversed by tetrahydrofolate. Unlike in the monkey liver enzyme, NAD+ and NADH gave considerable protection against this inhibition. The sheep liver enzyme differs significantly in its kinetic and regulatory properties from the serine hydroxymethyltransferases isolated from other sources.     

Regulation of monkey liver serine hydroxymethyltransferase by nicotinamide nucleotide.

The positive homotropic binding of tetrahydrofolate to monkey liver serine hydroxymethyltransferase was abolished on preincubating the enzyme with NADH and NADPH. NAD+ was a negative heterotropic effector, whereas NADP+ was without effect. The allosteric effects of nicotinamide nucleotides on the serine hydroxymethyltransferase, reported for the first time, lead to a better understanding of the regulation of the metabolic interconversion of folate coenzymes.     

Allosteric serine hydroxymethyltransferase from monkey liver: Temperature induced conformational transitions

The homogeneous serine hydroxymethyltransferase from monkey liver was optimally activate at 60°C and the Arrhenius plot for the enzyme was nonlinear with a break at 15°C. The monkey liver enzyme showed high thermal stability of 62°C, as monitored by circular dichroism at 222 nm, absorbance at 280 nm and enzyme activity. The enzyme exhibited a sharp co-operative thermal transition in the range of 50°–70°(T _m= 65°C), as monitored by circular dichroism. L-Serine protected the enzyme against both thermal inactivation and thermal disruption of the secondary structure. The homotropic interactions of tetrahydrofolate with the enzyme was abolished at high temperatures (at 70°C, the Hill coefficient value was 1.0). A plot ofh values vs. assay temperature of tetrahydrofolate saturation experiments, showed the presence of an intermediate conformer with anh value of 1.7 in the temperature range of 45°–60°C. Inclusion of a heat denaturation step in the scheme employed for the purification of serine hydroxymethyltransferase resulted in the loss of cooperative interactions with tetrahydrofolate. The temperature effects on the serine hydroxylmethyltransferase, reported for the first time, lead to a better understanding of the heat induced alterations in conformation and activity for this oligomeric protein.     

Inhibition of monkey liver serine hydroxymethyltransferase by Cibacron Blue 3G-A.

Cibacron Blue 3G-A inhibited monkey liver serine hydroxymethyltransferase competitively with respect to tetrahydrofolate and non-competitively with respect to L-serine. NADH, a positive heterotropic effector, failed to protect the enzymes against inhibition by the dye and was unable to desorb the enzyme from Blue Sepharose CL-6B gel matrix. The binding of the dye to the free enzyme was confirmed by changes in the dye absorption spectrum. The results indicate that the dye probably binds at the tetrahydrofolate-binding domain of the enzyme, rather than at the 'dinucleotide fold'.     

Disentangling the web of allosteric communication in a homotetramer: heterotropic activation in phosphofructokinase from Escherichia coli

Phosphofructokinase from Escherichia coli (EcPFK) is a homotetramer with four active sites and four allosteric sites. Understanding the allosteric activation of EcPFK by MgADP has been complicated by the complex web of possible interactions, including active site homotropic interactions, allosteric site homotropic interactions, and heterotropic interactions between active and allosteric sites. The current work has simplified this web of possible interactions to a series of single heterotropic interactions by forming and isolating hybrid tetramers. Each of the four unique heterotropic interactions have independently been isolated and compared to a control that has all four of the unique heterotropic interactions. If the interactions are labeled with the distances between interacting ligands, the 45-A interaction contributes 20% +/- 1%, the 33-A interaction contributes 34% +/- 1%, the 30-A interaction contributes 21% +/- 1%, and the 23-A interaction contributes 25% +/- 1% with respect to the total free energy of MgADP/fructose 6-phosphate (Fru-6-P) activation in the control. The free energies of the isolated interactions sum to 100% +/- 2% of the total. Therefore, the four unique interactions are all contributors to activation, are nonequivalent, and are additive.     

Structure-function relationship in allosteric aspartate carbamoyltransferase from Escherichia coli. II. Involvement of the C-terminal region of the regulatory chain in homotropic and heterotropic interactions

The modified aspartate transcarbamylase (ATCase) encoded by the transducing phage described by Cunin et al. has been purified to homogeneity. In this altered form of enzyme (pAR5-ATCase) the last eight amino acids of the C-terminal end of the regulatory chains are replaced by a sequence of six amino acids coded for by the lambda DNA. This modification has very informative consequences on the allosteric properties of ATCase. pAR5-ATCase lacks the homotropic co-operative interactions between the catalytic sites for aspartate binding and is "frozen" in the R state. In addition, this altered form of enzyme is insensitive to the physiological feedback inhibitor CTP, in spite of the fact that this nucleotide binds normally to the regulatory sites. Conversely, pAR5-ATCase is fully sensitive to the activator ATP. However, this activation is limited to the extent of the previously described "primary effect" as expected from an ATCase form "frozen" in the R state. These results emphasize the importance of the three-dimensional structure of the C-terminal region of the regulatory chains for both homotropic and heterotropic interactions. In addition, they indicate that the primary effects of CTP and ATP involve different features of the regulatory chain-catalytic chain interaction area.     

Specific suppression of heterotropic interactions in phosphofructokinase by the mutation of leucine 178 into tryptophan

The leucine residue at position 178 in the allosteric phosphofructokinase from Escherichia coli has been changed into a tryptophan residue by oligonucleotide-directed mutagenesis. The modified enzyme has been purified to homogeneity, and its enzymatic properties show that this single mutation suppresses the heterotropic interactions without affecting the homotropic ones. The mutant has the same saturation curve by fructose 6-phosphate as the wild type, showing that its active site binds this substrate with the same affinity and cooperativity. The regulatory site of the mutant enzyme can bind the effectors, the activator GDP, or the inhibitor phosphoenolpyruvate, as measured by protection against irreversible thermal denaturation. However, the binding of either effector does no longer influence the activity. This specific suppression of the coupling between the regulatory and active sites is not predicted by the concerted model which postulates that the same structural transition between two states R and T is responsible for both homotropic and heterotropic interactions. Leu-178 belongs to neither the active nor the regulatory site but appears as an important residue in the conformational change(s) involved in the regulation by allosteric effectors.     

Evidence that tetrahydrofolate does not bind to serine hydroxymethyltransferase with positive homotropic cooperativity

Previous experiments suggesting that tetrahydrofolate binds to serine hydroxymethyltransferase with positive homotropic cooperativity have been reinvestigated. Our results show that the sigmoid-shaped tetrahydrofolate saturation curve, previously obtained by several other investigators, is due to the instability of tetrahydrofolate in the assay solution. Using a different assay method, we have shown that tetrahydrofolate gives a hyperbolic saturation curve with serine hydroxymethyltransferase. We could find no evidence, as suggested by other investigators, that heating the enzyme during purification destroyed its allosteric properties or that NADH binds to the enzyme as an allosteric effector. Evidence is presented that the loss of tetrahydrofolate during the assay period is due to oxidation by dissolved molecular oxygen.     

An engineered liver glycogen phosphorylase with AMP allosteric activation

Liver and muscle glycogen phosphorylases, which are products of distinct genes, are both activated by covalent phosphorylation, but in the unphosphorylated (b) state, only the muscle isozyme is efficiently activated by the allosteric activator AMP. The different responsiveness of the phosphorylase isozymes to allosteric ligands is important for the maintenance of tissue and whole body glucose homeostasis. In an attempt to understand the structural determinants of differential sensitivity of the muscle and liver isozymes to AMP, we have developed a bacterial expression system for the liver enzyme, allowing native and engineered proteins to be expressed and characterized. Engineering of the single amino acid substitutions Thr48Pro, Met197Thr and the double mutant Thr48Pro, Met197Thr in liver phosphorylase, and Pro48Thr in muscle phosphorylase, did not qualitatively change the response of the two isozymes to AMP. These sites had previously been implicated in the configuration of the AMP binding site. However, when nine amino acids among the first 48 in liver phosphorylase were replaced with the corresponding muscle phosphorylase residues (L1M2-48L49-846), the engineered liver enzyme was activated by AMP to a higher maximal activity than native liver phosphorylase. Interestingly, the homotropic cooperativity of AMP binding was unchanged in the engineered phosphorylase b protein, and heterotropic cooperativity between the glucose-1-phosphate and AMP sites was only slightly enhanced. The native liver, native muscle and L1M2-48L49-846 phosphorylases were converted to the a form by treatment with purified phosphorylase kinase; the maximal activity of the chimeric a enzyme was greater than the native liver a enzyme and approached that of muscle phosphorylase a. From these results we suggest that tissue-specific phosphorylase isozymes have evolved a complex mechanism in which the N-terminal 48 amino acids modulate intrinsic activity (Vmax), probably by affecting subunit interactions, and other, as yet undefined regions specify the allosteric interactions with ligands and substrates.     

Different amino acid substitutions at the same position in the nucleotide-binding site of aspartate transcarbamoylase have diverse effects on the allosteric properties of the enzyme

Site-directed mutagenesis was used to determine how the allosteric properties of aspartate transcarbamoylase (ATCase) are affected by amino acid replacements in the nucleotide binding region of the regulatory polypeptide chains. Amino acid substitutions were made for both Lys-60 and Lys-94 in the regulatory chain since those residues have been implicated by x-ray diffraction studies, chemical modification experiments, and site-directed mutagenesis as playing a role in binding CTP and ATP. Lys-60 was replaced by His, Arg, Gln, and Ala, and Lys-94 was changed to His. These mutant forms of ATCase exhibit bewildering changes in the allosteric properties compared to the wild-type enzyme as well as altered affinities for the nucleotide effectors. The enzyme containing His-60 lacks both homotropic and heterotropic effects and exhibits no detectable binding of nucleotides. In contrast, the holoenzymes containing either Gln-60 or Arg-60 retain both homotropic and heterotropic effects. Replacement of Lys-60 by Ala yields a derivative exhibiting altered heterotropic effects involving insensitivity to CTP and activation by ATP. The mutant enzyme containing His-94 in place of Lys exhibits cooperativity with reduced affinity for nucleotides. The multiple substitutions at Lys-60 in the nucleotide binding region of the regulatory chains of ATCase demonstrate that different amino acids in the same location can alter indirectly the delicate balance of interactions responsible for the allosteric properties of ATCase. The studies show that it is hazardous and frequently unwarranted from single amino acid replacements of a specific residue to attribute to that residue the properties observed for the wild-type enzyme.     

Escherichia coli aspartate transcarbamoylase: the molecular basis for a concerted allosteric transition

Aspartate transcarbamoylase from Escherichia coli has become a model system for the study of both homotropic and heterotropic interactions in proteins. Analysis of the X-ray structures of the enzyme in the absence and presence of substrates and substrate analogs has revealed sets of interactions that appear to stabilize either the 'T' or the 'R' states of the enzyme. Site-specific mutagenesis has been used to test which of these interactions are functionally important. By combining the structural data from X-ray crystallography, and the functional data from site-specific mutagenesis a model is proposed for homotropic cooperativity in aspartate transcarbamoylase that suggests that the allosteric transition occurs in a concerted fashion.     

Studies on medium-chain fatty acyl-coenzyme A synthetase. Enzyme fraction I: mechanism of reaction and allosteric properties

1. The mechanism of butyrate activation catalysed by an enzyme fraction derived from ox liver particles (fraction I; Bar-Tana, Rose & Shapiro, 1968) was studied by an analysis of the initial-velocity pattern of the overall reaction and found to conform to the Bi Uni Uni Bi Ping Pong model (Cleland, 1963a,b,c) in agreement with the reaction scheme proposed by Berg (1956). 2. A homotropic co-operative effect was exerted by CoA on fraction I, whereas ATP and AMP functioned as heterotropic co-operative ligands with respect to butyryl-AMP-dependent CoA disappearance. On the other hand, PP(i) and butyryl-CoA showed antagonistic heterotropic effects when tested under similar conditions. With respect to the overall reaction CoA and ATP could be shown to function as co-operative homotropic modifiers. 3. Two interchangeable conformational states of the enzyme are therefore presumed to exist, state R, having a higher affinity for CoA and ATP and thus preferentially catalysing butyryl-AMP-dependent CoA disappearance (partial reaction b), and state T, favoured by the presence of PP(i), catalysing the formation of ATP from butyryl-AMP and PP(i) (partial reaction a) with greater efficiency. 4. These findings serve to explain the opposite effects of ATP on the partial reactions, as well as the inhibition by CoA and ATP of ATP formation (reaction a) and by PP(i) of the butyryl-AMP-dependent CoA disappearance (reaction b) (Bar-Tana et al. 1968). 5. The possible analogy of these observations to amino acid-activating and other similar systems is discussed.     

Plant aspartate transcarbamylase: an affinity chromatographic method for the purification of the enzyme from germinated seedings.

A simple and rapid affinity chromatographic method for the isolation of aspartate transcarbamylase from germinated seedlings of mung bean (Phaseolus aureus) was developed. A partially purified preparation of the enzyme was chromatographed on an affinity column containing aspartate linked to CNBr-activated Sepharose 4B. Aspartate transcarbamylase was specifically eluted from the column with 10 mm aspartate or 0.5 m KCl. The enzyme migrated as a single sharp band during disc electrophoresis at pH 8.6 on polyacrylamide gels. Electrophoresis of the sodium dodecyl sulfate-treated enzyme showed two distinct protein bands, suggesting that the mung bean aspartate transcarbamylase was made up of nonidentical subunits. Like the enzyme purified by conventional procedures, this enzyme preparation also exhibited positive homotropic interactions with carbamyl phosphate and negative heterotropic interactions with UMP. This method was extended to the purification of aspartate transcarbamylase from Lathyrus sativus, Eleucine coracona, and Trigonella foenum graecum.     

Discrimination between nucleotide effector responses of aspartate transcarbamoylase due to a single site substitution in the allosteric binding site

The substitution of alanine for lysine at position 56 of the regulatory polypeptide of aspartate transcarbamoylase affected both homotropic and heterotropic characteristics. In the absence of effectors, the ALAr56-substituted holoenzyme lost the homotropic cooperativity observed for aspartate in the wild-type holoenzyme. Under conditions of allosteric inhibition in the presence of 2mM CTP, the cooperative character of ATCase was restored, and the Hill coefficient increased from 1.0 to 1.7. In contrast to the native enzyme, the altered enzyme did not respond to ATP; however, ATP could still bind to the enzyme as demonstrated by its direct competition with CTP. Furthermore, the recently observed CTP-UTP synergism of the wild-type enzyme was not detectable. The site-directed mutant enzyme could not be activated by low levels of the bisubstrate analogue, N-(phosphonacetyl)-L-aspartate, and the rate of association of pHMB with the cysteine residues located at the interface of the catalytic and regulatory chains was slightly altered. These characteristics suggested that the mutant holoenzyme assumed a relaxed (or abnormal T state) conformation. Thus, this single substitution differentially affected the heterotropic responses to the various allosteric effectors of ATCase and eliminated the homotropic characteristics in response to aspartate in the absence of CTP.     

Threonine-sensitive homoserine dehydrogenase and aspartokinase activities of Escherichia coli K12. Kinetic and spectroscopic effects upon binding of serine and threonine.

The two threonine-sensitive activities aspartokinase and homoserine dehydrogenase are inhibited by L-serine. The inhibition of the aspartokinase by L-serine displays homotropic cooperative effects and is competitive versus aspartate. The inhibition by L-serine of the homoserine dehydrogenase displays Michaelis-Menten kinetics which are of a competitive nature versus homoserine. Characteristic effects of L-serine on the protein include a perturbation of its absorption and fluorescence spectra, with an increase in the fluorescence of the protein-NADPH complex. L-serine shifts the allosteric equilibrium of the protein to a "T-like" conformation to which L-threonine binds noncooperatively. L-Serine, a threonine analog, is not capable, as the physiological effector, of inducing a complete R to T transition of the enzyme; the aspartokinase globules show a cooperative conformation change upon serine binding, but this conformation change is not found in the homoserine dehydrogenase globules.     

The contribution of individual interchain interactions to the stabilization of the T and R states of Escherichia coli aspartate transcarbamoylase

Stabilization of the T and R allosteric states of Escherichia coli aspartate transcarbamoylase is governed by specific intra- and interchain interactions. The six interchain interactions between Glu-239 in one catalytic chain of one catalytic trimer with both Lys-164 and Tyr-165 of a different catalytic chain in the other catalytic trimer have been shown to be involved in the stabilization of the T state. In this study a series of hybrid versions of aspartate transcarbamoylase was studied to determine the minimum number of these Glu-239 interactions necessary to maintain homotropic cooperativity and the T allosteric state. Hybrids with zero, one, and two Glu-239 stabilizing interactions do not exhibit cooperativity, whereas the hybrids with three or more Glu-239 stabilizing interactions exhibit cooperativity. The hybrid enzymes with one or more of the Glu-239 stabilizing interactions also exhibit heterotropic interactions. Two hybrids with three Glu-239 stabilizing interactions, in different geometric relationships, had identical properties. From this and previous studies, it is concluded that the 239 stabilizing interactions play a critical role in the manifestation of homotropic cooperativity in aspartate transcarbamoylase by the stabilization of the T state of the enzyme. As substrate binding energy is utilized, more and more of the T state stabilizing interactions are relaxed, and finally the enzyme shifts to the R state. In the case of the Glu-239 stabilizing interactions more than three of the interactions must be broken before the enzyme shifts to the R state. The interactions between the catalytic and regulatory chains and between the two catalytic trimers of aspartate transcarbamoylase provide a global set of interlocking interactions that stabilize the T and R states of the enzyme. The substrate-induced local conformational changes observed in the structure of the isolated catalytic subunit drive the quaternary T to R transition of aspartate transcarbamoylase and functionally induced homotropic cooperativity.     

Global allostery model of hemoglobin. Modulation of O(2) affinity,cooperativity, and Bohr effect by heterotropic allosteric effectors

The O(2) equilibria of human adult hemoglobin have been measured in a wide range of solution conditions in the presence and absence of various allosteric effectors in order to determine how far hemoglobin can modulate its O(2) affinity. The O(2) affinity, cooperative behavior, and the Bohr effect of hemoglobin are modulated principally by tertiary structural changes, which are induced by its interactions with heterotropic allosteric effectors. In their absence, hemoglobin is a high affinity, moderately cooperative O(2) carrier of limited functional flexibility, the behaviors of which are regulated by the homotropic, O(2)-linked T/R quaternary structural transition of the Monod-Wyman-Changeux/Perutz model. However, the interactions with allosteric effectors provide such "inert" hemoglobin unprecedented magnitudes of functional diversities not only of physiological relevance but also of extreme nature, by which hemoglobin can behave energetically beyond what can be explained by the Monod-Wyman-Changeux/Perutz model. Thus, the heterotropic effector-linked tertiary structural changes rather than the homotropic ligation-linked T/R quaternary structural transition are energetically more significant and primarily responsible for modulation of functions of hemoglobin.     

Isolation of a single activating allosteric interaction in phosphofructokinase from Escherichia coli

Escherichia coli phosphofructokinase 1 (EcPFK) is a homotetramer with four active and four allosteric sites. Understanding of the structural basis of allosteric activation of EcPFK by MgADP is complicated by the multiplicity of binding sites. To isolate a single heterotropic allosteric interaction, hybrid tetramers were formed between wild-type and mutant EcPFK subunits in which the binding sites of the mutant subunits have decreased affinity for their respective ligands. The 1:3 (wild-type:mutant) hybrid that contained only one native active site and one native allosteric site was isolated. The affinity for the substrate fructose-6-phosphate (Fru-6-P) of a single wild-type active site is greatly decreased over that displayed by the wild-type tetramer due to the lack of homotropic activation. The free energy of activation by MgADP for this heterotropic interaction is -0.58 kcal/mol at 8.5 degrees C. This compares to -2.87 kcal/mol for a hybrid with no homotropic coupling but all four unique heterotropic interactions. Therefore, the isolated interaction contributes 20% of the total heterotropic coupling. By comparison, wild-type EcPFK exhibits a coupling free energy between Fru-6-P and MgADP of -1.56 kcal/mol under these conditions, indicating that the effects of MgADP are diminished by a homotropic activation equal to -1.3 kcal/mol. These data are not consistent with a concerted allosteric mechanism.     

Kinetic characterization of glycogen phosphorylase B from skeletal muscle of the mullet Liza ramada

1. Glycogen phosphorylase purified from muscle of mullet (Liza ramada) has been kinetically characterized. 2. Kinetic analysis for the substrates glucose-1-P and glycogen showed no homotropic co-operativity. AMP exhibited only a slight homotropic co-operative behaviour, although it caused a decrease in the Km for glucose-1-P. 3. Glucose, ATP and glucose-6-P behaved as phosphorylase b inhibitors. Kinetic analysis of the inhibition showed the characteristic heterotropic effect both for the substrate glucose-1-P and the activator AMP. 4. However, glucose-6-P, which enhances the co-operativity between AMP molecules, lost its heterotropic effect on the glucose-1-P saturation curve.     

Three of the six possible intersubunit stabilizing interactions involving Glu-239 are sufficient for restoration of the homotropic and heterotropic properties of Escherichia coli aspartate transcarbamoylase

A hybrid version of Escherichia coli aspartate transcarbamoylase was investigated in which one catalytic subunit has the wild-type sequence, and the other catalytic subunit has Glu-239 replaced by Gln. Since Glu-239 is involved in intersubunit interactions, this hybrid could be used to evaluate the extent to which T state stabilization is required for homotropic cooperativity and for heterotropic effects. Reconstitution of the hybrid holoenzyme (two different catalytic subunits with three wild-type regulatory subunits) was followed by separation of the mixture by anion-exchange chromatography. To make possible the resolution of the three holoenzyme species formed by the reconstitution, the charge of one of the catalytic subunits was altered by the addition of six aspartic acid residues to the C terminus of each of the catalytic chains (AT-C catalytic subunit). Control experiments indicated that the AT-C catalytic subunit as well as the holoenzyme formed with AT-C and wild-type regulatory subunits had essentially the same homotropic and heterotropic properties as the native catalytic subunit and holoenzyme, indicating that the addition of the aspartate tail did not influence the function of either enzyme. The control reconstituted holoenzyme, in which both catalytic subunits have Glu-239 replaced by Gln, exhibited no cooperativity, an enhanced affinity for aspartate, and essentially no heterotropic response identical to the enzyme isolated without reconstitution. The hybrid containing one normal and one mutant catalytic subunit exhibited homotropic cooperativity with a Hill coefficient of 1.4 and responded to the nucleotide effectors at about 50% of the level of the wild-type enzyme. Small angle x-ray scattering experiments with the hybrid enzyme indicated that in the absence of ligands it was structurally similar, but not identical, to the T state of the wild-type enzyme. In contrast to the wild-type enzyme, addition of carbamoyl phosphate induced a significant alteration in the scattering pattern, whereas the bisubstrate analog N-phosphonoacetyl-L-aspartate induced a significant change in the scattering pattern indicating the transition to the R-structural state. These data indicate that in the hybrid enzyme only three of the usual six interchain interactions involving Glu-239 are sufficient to stabilize the enzyme in a low affinity, low activity state and allow an allosteric transition to occur.