Biomedical subjects

A Mozzarelli

Publications and source records attributed to A Mozzarelli.

At least 19 recordsLinked to original sources

PubMed2001

Mutational effects at the subunit interfaces of human hemoglobin: evidence for a unique sensitivity of the T quaternary state to changes in the hinge region of the alpha 1 beta 2 interface.

A set of variant human hemoglobins, each with an Ala or Gly substitution at a single residue, has been prepared, and the kinetics of their reactions with carbon monoxide have been measured. This reaction is rate-limited by the binding of the first CO to the deoxygenated T state of the protein. The magnitudes of the effects of the mutations on CO combination vary widely, and, with the exception of beta Y145, the residues with the most significant effects on these kinetics are found in the hinge region of the alpha 1 beta 2 interface. Mixed-metal hybrids, with zinc protoporphyrin IX in place of heme on both alpha or both beta subunits, were prepared for beta W37E, beta W37A, alpha Y140G, and alpha Y140A, hinge region variants causing large kinetic changes, and for beta Y145G. Such hybrids permit measurements of the kinetics of CO binding to only the heme-containing alpha or beta subunits within the unliganded hemoglobin tetramer. Mutations at beta 37 and alpha 140 have global effects on the T state, increasing the rates of CO binding to both types of subunits. Mutation of beta Y145 has a large effect on the beta subunits in the deoxygenated T state, but very little effect on the alpha subunits. Oxygen equilibria measurements on the crystalline T state of beta W37E also indicate large affinity increases in both subunits of this variant. The overall oxygen equilibria of the variant hemoglobins in solution are sensitive to numerous variables besides the properties of the deoxygenated T state. In contrast to CO combination kinetics, the residues whose alterations cause the largest changes in overall oxygen equilibria in solution are scattered seemingly randomly within the alpha 1 beta 2 interface.

Amino Acid Substitution↗

PubMed2001

Allosteric communication of tryptophan synthase. Functional and regulatory properties of the beta S178P mutant.

The alpha(2)beta(2) tryptophan synthase complex is a model enzyme for understanding allosteric regulation. We report the functional and regulatory properties of the betaS178P mutant. Ser-178 is located at the end of helix 6 of the beta subunit, belonging to the domain involved in intersubunit signaling. The carbonyl group of betaSer-178 is hydrogen bonded to Gly-181 of loop 6 of the alpha subunit only when alpha subunit ligands are bound. An analysis by molecular modeling of the structural effects caused by the betaS178P mutation suggests that the hydrogen bond involving alphaGly-181 is disrupted as a result of localized structural perturbations. The ratio of alpha to beta subunit concentrations was calculated to be 0.7, as for the wild type, indicating the maintenance of a tight alpha-beta complex. Both the activity of the alpha subunit and the inhibitory effect of the alpha subunit ligands indole-3-acetylglycine and d,l-alpha-glycerol-3-phosphate were found to be the same for the mutant and wild type enzyme, whereas the beta subunit activity of the mutant exhibited a 2-fold decrease. In striking contrast to that observed for the wild type, the allosteric effectors indole-3-acetylglycine and d,l-alpha-glycerol-3-phosphate do not affect the beta activity. Accordingly, the distribution of l-serine intermediates at the beta-site, dominated by the alpha-aminoacrylate, is only slightly influenced by alpha subunit ligands. Binding of sodium ions is weaker in the mutant than in the wild type and leads to a limited increase of the amount of the external aldimine intermediate, even at high pH, whereas binding of cesium ions exhibits the same affinity and effects as in the wild type, leading to an increase of the alpha-aminoacrylate tautomer absorbing at 450 nm. Crystals of the betaS178P mutant were grown, and their functional and regulatory properties were investigated by polarized absorption microspectrophotometry. These findings indicate that (i) the reciprocal activation of the alpha and beta activity in the alpha2beta2 complex with respect to the isolated subunits results from interactions that involve residues different from betaSer-178 and (ii) betaSer-178 is a critical residue in ligand-triggered signals between alpha and beta active sites.

Allosteric Regulation↗

PubMed2001

Functional properties of the active core of human cystathionine beta-synthase crystals.

Human cystathionine beta-synthase is a pyridoxal 5'-phosphate enzyme containing a heme binding domain and an S-adenosyl-l-methionine regulatory site. We have investigated by single crystal microspectrophotometry the functional properties of a mutant lacking the S-adenosylmethionine binding domain. Polarized absorption spectra indicate that oxidized and reduced hemes are reversibly formed. Exposure of the reduced form of enzyme crystals to carbon monoxide led to the complete release of the heme moiety. This process, which takes place reversibly and without apparent crystal damage, facilitates the preparation of a heme-free human enzyme. The heme-free enzyme crystals exhibited polarized absorption spectra typical of a pyridoxal 5'-phosphate-dependent protein. The exposure of these crystals to increasing concentrations of the natural substrate l-serine readily led to the formation of the key catalytic intermediate alpha-aminoacrylate. The dissociation constant of l-serine was found to be 6 mm, close to that determined in solution. The amount of the alpha-aminoacrylate Schiff base formed in the presence of l-serine was pH independent between 6 and 9. However, the rate of the disappearance of the alpha-aminoacrylate, likely forming pyruvate and ammonia, was found to increase at pH values higher than 8. Finally, in the presence of homocysteine the alpha-aminoacrylate-enzyme absorption band readily disappears with the concomitant formation of the absorption band of the internal aldimine, indicating that cystathionine beta-synthase crystals catalyze both beta-elimination and beta-replacement reactions. Taken together, these findings demonstrate that the heme moiety is not directly involved in the condensation reaction catalyzed by cystathionine beta-synthase.

Binding Sites↗

PubMed2001

Molecular heterogeneity of O-acetylserine sulfhydrylase by two-photon excited fluorescence fluctuation spectroscopy.

O-acetylserine sulfhydrylase, a homo-dimeric enzyme from Salmonella typhimurium, covalently binds one pyridoxal 5'-phosphate molecule per subunit as a fluorescent coenzyme. Different tautomers of the Schiff base between the coenzyme and lysine 41 generate structured absorption and fluorescence spectra upon one-photon excitation. We investigated the protein population heterogeneity by fluorescence correlation spectroscopy and lifetime techniques upon two-photon excitation. We sampled the fluorescence intensity from a small number of molecules (approximately 10) and analyzed the distribution of photon counts to separately determine the number and the fluorescence brightness of excited protein molecules. The changes in the average number of molecules and in the fluorescence brightness with the excitation wavelength indicate the presence of at least two fluorescent species, with two-photon excitation maxima at 660 and 800 nm. These species have been identified as the enolimine and ketoenamine tautomers of the protein-coenzyme internal aldimine. Their relative abundance is estimated to be 4:1, whereas the ratio of their two-photon cross sections is reversed with respect to the single-photon excitation case. Consistent results are obtained from the measurement of the lifetime decays, which are sensitive to the excited-state heterogeneity. At least two components were detected, with lifetimes of approximately 2.5 and 0.5 ns. The lifetimes are very close to the values measured in bulk solutions upon one-photon excitation and attributed to the ketoenamine tautomer and to a dipolar species formed upon proton dissociation in the excited state.

Biochemistry↗

PubMed2001

Site-directed mutations of human hemoglobin at residue 35beta: a residue at the intersection of the alpha1beta1, alpha1beta2, and alpha1alpha2 interfaces.

Because Tyr35beta is located at the convergence of the alpha1beta1, alpha1beta2, and alpha1alpha2 interfaces in deoxyhemoglobin, it can be argued that mutations at this position may result in large changes in the functional properties of hemoglobin. However, only small mutation-induced changes in functional and structural properties are found for the recombinant hemoglobins betaY35F and betaY35A. Oxygen equilibrium-binding studies in solution, which measure the overall oxygen affinity (the p50) and the overall cooperativity (the Hill coefficient) of a hemoglobin solution, show that removing the phenolic hydroxyl group of Tyr35beta results in small decreases in oxygen affinity and cooperativity. In contrast, removing the entire phenolic ring results in a fourfold increase in oxygen affinity and no significant change in cooperativity. The kinetics of carbon monoxide (CO) combination in solution and the oxygen-binding properties of these variants in deoxy crystals, which measure the oxygen affinity and cooperativity of just the T quaternary structure, show that the ligand affinity of the T quaternary structure decreases in betaY35F and increases in betaY35A. The kinetics of CO rebinding following flash photolysis, which provides a measure of the dissociation of the liganded hemoglobin tetramer, indicates that the stability of the liganded hemoglobin tetramer is not altered in betaY35F or betaY35A. X-ray crystal structures of deoxy betaY35F and betaY35A are highly isomorphous with the structure of wild-type deoxyhemoglobin. The betaY35F mutation repositions the carboxyl group of Asp126alpha1 so that it may form a more favorable interaction with the guanidinium group of Arg141alpha2. The betaY35A mutation results in increased mobility of the Arg141alpha side chain, implying that the interactions between Asp126alpha1 and Arg141alpha2 are weakened. Therefore, the changes in the functional properties of these 35beta mutants appear to correlate with subtle structural differences at the C terminus of the alpha-subunit.

Amino Acid Substitution↗

PubMed2001

High and low oxygen affinity conformations of T state hemoglobin.

To understand the interplay between tertiary and quaternary transitions associated with hemoglobin function and regulation, oxygen binding curves were obtained for hemoglobin A fixed in the T quaternary state by encapsulation in wet porous silica gels. At pH 7.0 and 15 degrees C, the oxygen pressure at half saturation (p50) was measured to be 12.4 +/- 0.2 and 139 +/- 4 torr for hemoglobin gels prepared in the absence and presence of the strong allosteric effectors inositol hexaphosphate and bezafibrate, respectively. Both values are in excellent agreement with those found for the binding of the first oxygen to hemoglobin in solution under similar experimental conditions. The corresponding Hill coefficients of hemoglobin gels were 0.94 +/- 0.02 and 0.93 +/- 0.03, indicating, in the frame of the Monod, Wyman, and Changeux model, that high and low oxygen-affinity tertiary T-state conformations have been isolated in a pure form. The values, slightly lower than unity, reflect the different oxygen affinity of alpha- and beta-hemes. Significantly, hemoglobin encapsulated in the presence of the weak effector phosphate led to gels that show intermediate oxygen affinity and Hill coefficients of 0.7 to 0.8. The heterogeneous oxygen binding results from the presence of a mixture of the high and low oxygen-affinity T states. The Bohr effect was measured for hemoglobin gels containing the pure conformations and found to be more pronounced for the high-affinity T state and almost absent for the low-affinity T state. These findings indicate that the functional properties of the T quaternary state result from the contribution of two distinct, interconverting conformations, characterized by a 10-fold difference in oxygen affinity and a different extent of tertiary Bohr effect. The very small degree of T-state cooperativity observed in solution and in the crystalline state might arise from a ligand-induced perturbation of the distribution between the high- and low-affinity T-state conformations.

Allosteric Site↗

PubMed2001

Oxygen binding by single red blood cells from the red-eared turtle Trachemys scripta.

Oxygen-binding properties of single red blood cells from the red-eared turtle Trachemys scripta were measured by microspectrophotometry to describe the variation in oxygen affinity of red blood cells and to gain insight into the distribution of functionally different hemoglobins among red blood cells. Methodologically, this study represents the first report on the cell-to-cell variation in oxygen-binding properties based on oxygen-binding curves of single vertebrate red blood cells. The cells differed significantly with respect to oxygen affinity. Mean oxygen pressure at half saturation of the cells in a blood sample was found to be 20.1 +/- 3.3 (SD) Torr. The distribution of oxygen affinities among red blood cells is unimodal, indicating that the two hemoglobins found in turtle blood are not segregated in distinct cells. Therefore, the functional interaction shown by these hemoglobins in vitro is likely to take place in vivo. The considerable variation in oxygen affinity between individual red blood cells calls for its incorporation in models of tissue oxygenation.

Adenosine Triphosphate↗

PubMed2001

Functional characterization of heme proteins encapsulated in wet nanoporous silica gels.

Myoglobin and hemoglobin were encapsulated in wet, nanoporous silica gels. A rigorous evaluation of the effect of the encapsulation on protein dynamics and function was carried out by measuring the kinetics of carbon monoxide rebinding after nanosecond laser flash-photolysis with transient absorption detection, and the oxygen affinity with absorption microspectrophotometry. The time course of carbon monoxide binding to myoglobin evidenced a strongly enhanced geminate recombination and a faster bimolecular rebinding with respect to solution, whereas T and R quaternary states of hemoglobin exhibited a geminate phase and a bimolecular binding rate very similar to those observed in solution. Oxygen affinity of T-state hemoglobin was found to be close to that observed for the binding of the first oxygen to T-state hemoglobin in solution. Results indicate that some conformational transitions are kinetically restricted, allowing to isolate distinct tertiary and quaternary states. This opens the way to their detailed functional characterization and application to biodevices.

Absorption↗

PubMed2000

Role of pyridoxal 5'-phosphate in the structural stabilization of O-acetylserine sulfhydrylase.

Proteins belonging to the superfamily of pyridoxal 5'-phosphate-dependent enzymes are currently classified into three functional groups and five distinct structural fold types. The variation within this enzyme group creates an ideal system to investigate the relationships among amino acid sequences, folding pathways, and enzymatic functions. The number of known three-dimensional structures of pyridoxal 5'-phosphate-dependent enzymes is rapidly increasing, but only for relatively few have the folding mechanisms been characterized in detail. The dimeric O-acetylserine sulfhydrylase from Salmonella typhimurium belongs to the beta-family and fold type II group. Here we report the guanidine hydrochloride-induced unfolding of the apo- and holoprotein, investigated using a variety of spectroscopic techniques. Data from absorption, fluorescence, circular dichroism, (31)P nuclear magnetic resonance, time-resolved fluorescence anisotropy, and photon correlation spectroscopy indicate that the O-acetylserine sulfhydrylase undergoes extensive disruption of native secondary and tertiary structure before monomerization. Also, we have observed that the holo-O-acetylserine sulfhydrylase exhibits a greater conformational stability than the apoenzyme form. The data are discussed in light of the fact that the role of the coenzyme in structural stabilization varies among the pyridoxal 5'-phosphate-dependent enzymes and does not seem to be linked to the particular enzyme fold type.

Cysteine Synthase↗

PubMed2000

HINT predictive analysis of binding between retinol binding protein and hydrophobic ligands.

The interaction between the retinol binding protein and four ligands was evaluated using HINT, a software based on experimental LogP values of individual atoms. A satisfactory correlation was found between the HINT scores and the experimental dissociation constants of three of the ligands, fenretinide, N-ethylretinamide and all-trans retinol, despite their hydrophobic nature. A prediction is made for the binding affinity of the fourth ligand, axerophtene, not yet determined in solution.

Algorithms↗

PubMed2000

Effect of pH and monovalent cations on the formation of quinonoid intermediates of the tryptophan synthase alpha(2)beta(2) complex in solution and in the crystal.

Quinonoid intermediates play a key role in the catalytic mechanism of pyridoxal 5'-phosphate-dependent enzymes. Whereas the structures of other pyridoxal 5'-phosphate-bound intermediates have been determined, the structure of a quinonoid species has not yet been reported. Here, we investigate factors controlling the accumulation and stability of quinonoids formed at the beta-active site of tryptophan synthase both in solution and the crystal. The quinonoids were obtained by reacting the alpha-aminoacrylate Schiff base with different nucleophiles, focusing mainly on the substrate analogs indoline and beta-mercaptoethanol. In solution, both monovalent cations (Cs(+) or Na(+)) and alkaline pH increase the apparent affinity of indoline and favor accumulation of the indoline quinonoid. A similar pH dependence is observed when beta-mercaptoethanol is used. As indoline and beta-mercaptoethanol exhibit very distinct ionization properties, this finding suggests that nucleophile binding and quinonoid stability are controlled by some ionizable protein residue(s). In the crystal, alkaline pH favors formation of the indoline quinonoid as in solution, but the effect of cations is markedly different. In the absence of monovalent metal ions the quinonoid species accumulates substantially, whereas in the presence of sodium ions the accumulation is modest, unless alpha-subunit ligands are also present. Alpha-subunit ligands not only favor the formation of the intermediate, but also reduce significantly its decay rate. These findings define experimental conditions suitable for the stabilization of the quinonoid species in the crystal, a critical prerequisite for the determination of the three-dimensional structure of this intermediate.

Alanine↗

PubMed2000

Novel allosteric effectors of the tryptophan synthase alpha(2)beta(2) complex identified by computer-assisted molecular modeling.

Tryptophan synthase is a pyridoxal 5'-phosphate-dependent alpha(2)beta(2) complex catalyzing the formation of L-tryptophan. The functional properties of one subunit are allosterically regulated by ligands of the other subunit. Molecules tailored for binding to the alpha-active site were designed using as a starting model the three-dimensional structure of the complex between the enzyme from Salmonella typhimurium and the substrate analog indole-3-propanol phosphate. On the basis of molecular dynamics simulations, indole-3-acetyl-X, where X is glycine, alanine, valine and aspartate, and a few other structurally related compounds were found to be good candidates for ligands of the alpha-subunit. The binding of the designed compounds to the alpha-active site was evaluated by measuring the inhibition of the alpha-reaction of the enzyme from Salmonella typhimurium. The inhibition constants were found to vary between 0.3 and 1.7 mM. These alpha-subunit ligands do not bind to the beta-subunit, as indicated by the absence of effects on the rate of the beta-reaction in the isolated beta(2) dimer. A small inhibitory effect on the activity of the alpha(2)beta(2) complex was caused by indole-3-acetyl-glycine and indole-3-acetyl-aspartate whereas a small stimulatory effect was caused by indole-3-acetamide. Furthermore, indole-3-acetyl-glycine, indole-3-acetyl-aspartate and indole-3-acetamide perturb the equilibrium of the catalytic intermediates formed at the beta-active site, stabilizing the alpha-aminoacrylate Schiff base. These results indicate that (i) indole-3-acetyl-glycine, indole-3-acetyl-aspartate and indole-3-acetamide bind to the alpha-subunit and act as allosteric effectors whereas indole-3-acetyl-valine and indole-3-acetyl-alanine only bind to the alpha-subunit, and (ii) the terminal phosphate present in the already known allosteric effectors of tryptophan synthase is not strictly required for the transmission of regulatory signals.

Allosteric Regulation↗

PubMed2000

Oxygen binding by alpha(Fe2+)2beta(Ni2+)2 hemoglobin crystals.

Oxygen binding by hemoglobin fixed in the T state either by crystallization or by encapsulation in silica gels is apparently noncooperative. However, cooperativity might be masked by different oxygen affinities of alpha and beta subunits. Metal hybrid hemoglobins, where the noniron metal does not bind oxygen, provide the opportunity to determine the oxygen affinities of alpha and beta hemes separately. Previous studies have characterized the oxygen binding by alpha(Ni2+)2beta(Fe2+)2 crystals. Here, we have determined the three-dimensional (3D) structure and oxygen binding of alpha(Fe2+)2beta(Ni2+)2 crystals grown from polyethylene glycol solutions. Polarized absorption spectra were recorded at different oxygen pressures with light polarized parallel either to the b or c crystal axis by single crystal microspectrophotometry. The oxygen pressures at 50% saturation (p50s) are 95 +/- 3 and 87 +/- 4 Torr along the b and c crystal axes, respectively, and the corresponding Hill coefficients are 0.96 +/- 0.06 and 0.90 +/- 0.03. Analysis of the binding curves, taking into account the different projections of the alpha hemes along the optical directions, indicates that the oxygen affinity of alpha1 hemes is 1.3-fold lower than alpha2 hemes. Inspection of the 3D structure suggests that this inequivalence may arise from packing interactions of the Hb tetramer within the monoclinic crystal lattice. A similar inequivalence was found for the beta subunits of alpha(Ni2+)2beta(Fe2+)2 crystals. The average oxygen affinity of the alpha subunits (p50 = 91 Torr) is about 1.2-fold higher than the beta subunits (p50 = 110 Torr). In the absence of cooperativity, this heterogeneity yields an oxygen binding curve of Hb A with a Hill coefficient of 0.999. Since the binding curves of Hb A crystals exhibit a Hill coefficient very close to unity, these findings indicate that oxygen binding by T-state hemoglobin is noncooperative, in keeping with the Monod, Wyman, and Changeux model.

Crystallization↗

PubMed1999

Anticooperative ligand binding properties of recombinant ferric Vitreoscilla homodimeric hemoglobin: a thermodynamic, kinetic and X-ray crystallographic study.

Thermodynamics and kinetics for cyanide, azide, thiocyanate and imidazole binding to recombinant ferric Vitreoscilla sp. homodimeric hemoglobin (Vitreoscilla Hb) have been determined at pH 6.4 and 7.0, and 20.0 degrees C, in solution and in the crystalline state. Moreover, the three-dimensional structures of the diligated thiocyanate and imidazole derivatives of recombinant ferric Vitreoscilla Hb have been determined by X-ray crystallography at 1.8 A (Rfactor=19.9%) and 2.1 A (Rfactor=23.8%) resolution, respectively. Ferric Vitreoscilla Hb displays an anticooperative ligand binding behaviour in solution. This very unusual feature can only be accounted for by assuming ligand-linked conformational changes in the monoligated species, which lead to the observed 300-fold decrease in the affinity of cyanide, azide, thiocyanate and imidazole for the monoligated ferric Vitreoscilla Hb with respect to that of the fully unligated homodimer. In the crystalline state, thermodynamics for azide and imidazole binding to ferric Vitreoscilla Hb may be described as a simple process with an overall ligand affinity for the homodimer corresponding to that for diligation in solution. These data suggest that the ligand-free homodimer, observed in the crystalline state, is constrained in a low affinity conformation whose ligand binding properties closely resemble those of the monoligated species in solution. From the kinetic viewpoint, anticooperativity is reflected by the 300-fold decrease of the second-order rate constant for cyanide and imidazole binding to the monoligated ferric Vitreoscilla Hb with respect to that for ligand association to the ligand-free homodimer in solution. On the other hand, values of the first-order rate constant for cyanide and imidazole dissociation from the diligated and monoligated derivatives of ferric Vitreoscilla Hb in solution are closely similar. As a whole, ligand binding and structural properties of ferric Vitreoscilla Hb appear to be unique among all Hbs investigated to date.

Crystallography, X-Ray↗

PubMed1999

Time-resolved fluorescence of O-acetylserine sulfhydrylase.

Static and time-resolved fluorescence of the internal aldimine of the pyridoxal 5'-phosphate (PLP)-dependent enzyme O-acetylserine sulfhydrylase (OASS) and those of free PLP, and the PLP-L-valine Schiff base have been measured to gain insight into the photophysics of PLP bound to OASS. Exciting at 330 nm, free coenzyme exhibits a band at 415 nm, whereas PLP-valine and OASS (also when excited at their absorbance maxima) exhibit a structured emission with a peak at 420 nm and shoulders at 490 and 530 nm. The emission bands at 420 and 490 nm are attributed to the enolimine and ketoenamine tautomers of the internal aldimine, respectively, while the 530 nm emission might arise from a dipolar species formed upon proton dissociation in the excited state. Time-resolved fluorescence of OASS (PLP-valine), excited at 412 nm (415 nm) and collected at lamda > 470 nm, indicates the presence of two components characterized by lifetimes (tau) of 0.6 (0.08) and 3.8 (1.55) ns with equal fractional intensity (f). In the presence of acetate the slow component dominates OASS emission with f of 0.98. Excitation at 350 nm as a function of emission wavelengths (400-560 nm) shows at least three components. The f of the slow component increases from 400 to 440 nm, then decreases, whereas the f of the intermediate and fast components behave in the opposite way. Results indicate that: (i) the fast component is associated with the emission at 530 nm; (ii) the slow component is associated with the emission at 420 nm; (iii) a fast additive component, characterized by a very short lifetime, is present on the blue side of the emission spectrum; (iv) the intermediate component results from overlapping contributions, including the emission of the band at 490 nm, that could not be resolved; (v) the increased emission at 490 nm, caused by acetate binding, is likely due to the stabilization of the ketoenamine tautomer induced by an increase in polarity of the active site microenvironment and/or a decrease in proton dissociation in the excited state; (vi) excitation at 330 nm, where the enolimine tautomer absorbs, leads to emission decays typical of the ketoenamine.

Acetates↗

PubMed1999

Is cooperative oxygen binding by hemoglobin really understood?

The enormous success of structural biology challenges the physical scientist. Can biophysical studies provide a truly deeper understanding of how a protein works than can be obtained from static structures and qualitative analysis of biochemical data? We address this question in a case study by presenting the key concepts and experimental results that have led to our current understanding of cooperative oxygen binding by hemoglobin, the paradigm of structure function relations in multisubunit proteins. We conclude that the underlying simplicity of the two-state allosteric mechanism could not have been demonstrated without novel physical experiments and a rigorous quantitative analysis.

Chemistry, Physical↗

PubMed1998

Plasticity of the tryptophan synthase active site probed by 31P NMR spectroscopy.

The functional properties of tryptophan synthase alpha2beta2 complex are modulated by a variety of allosteric effectors, including pH, monovalent cations, and alpha-subunit ligands. The dynamic properties of the beta-active site were probed by 31P NMR spectroscopy of the enzyme-bound coenzyme pyridoxal 5'-phosphate. The 31P NMR signal of the cofactor phosphate of the internal aldimine exhibits a single peak at 3.73 ppm with a line width of 12 Hz. In the presence of saturating concentrations of sodium ions, the 31P signal shifts to 3.97 ppm concomitant with a change in line width to 35 Hz. The latter indicates that sodium ions decrease the conformational flexibility of the coenzyme. In the absence of ions, lowering pH leads to the appearance of a second peak at 4.11 ppm, the intensity of which decreases in the presence of cesium ions. Addition of L-serine in the presence of sodium ions leads to the formation of the external aldimine, the first metastable catalytic intermediate. The 31P signal does not change its position, but a change in line width from 35 to 5 Hz is observed, revealing that this species is characterized by a considerable degree of rotational freedom around the coenzyme C-O bond. In the presence of L-serine and either cesium ions or the allosteric effector indole-3-acetylglycine, the accumulation of the second catalytic intermediate, alpha-aminoacrylate, is observed. The 31P signal is centered at 3.73 ppm with a line width of 5 Hz, indicating that the phosphate group of the coenzyme in the external aldimine and the alpha-aminoacrylate exhibits the same flexibility but a slightly different state of ionization. Because the alpha-aminoacrylate intermediate but not the external aldimine triggers the allosteric signal to the alpha-subunit, other portions of the beta-active site modify their dynamic properties in response to the progress of the catalytic process. A narrow line width was also observed for the quinonoid species formed by nucleophilic attack of indoline to the alpha-aminoacrylate. The 31P signal moves downfield to 4.2 ppm, indicating a possible change of the ionization state of the phosphate group. Thus, the modification of either the ionization state of the coenzyme phosphate or its flexibility or both are, at least in part, responsible for the conformational events that accompany the catalytic process.

Alanine↗

PubMed1998

Allosteric mechanism of haemoglobin: rupture of salt-bridges raises the oxygen affinity of the T-structure.

The T-structure of human haemoglobin is linked by salt-bridges between its four subunits, formed by the C-terminal arginine residues of the alpha-subunits and the C-terminal histidine residues of the beta-subunits. In the R-structure, these salt-bridges are absent. The oxygen affinity of the T-structure is lower than that of the R-structure by the equivalent of 3.5 kcal/mol haem. This difference has been attributed to the constraints imposed upon the T-structure by the salt-bridges, which were thought to hinder the changes in tertiary structure needed for firm oxygen binding. We have subjected this postulate to a rigorous test by measuring the oxygen equilibria of T-state crystals of an abnormal human haemoglobin in which the C-terminal histidine residues of the beta-chains are replaced by leucine residues. This replacement removes the salt-bridges from the histidine imidazole groups to the neighbouring aspartate residues. The crystals have an oxygen affinity about three times greater than that of crystals of normal haemoglobin. Hill's coefficient is close to unity. The oxygen affinity is unaffected by pH, chloride or the allosteric effector bezafibrate. Equilibrium curves determined by single crystal microspectrophometry using light polarised parallel and normal to the crystallographic a-axis show no significant difference between the oxygen affinities of alpha and beta-haems. Our results show that rupture of salt-bridges raises the oxygen affinity of the T-structure even when this is clamped firmly by the crystal lattice.

Allosteric Regulation↗