Biomedical subjects

A Mozzarelli

Publications and source records attributed to A Mozzarelli.

At least 19 recordsLinked to original sources

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↗

PubMed1998

Cryocrystallography and microspectrophotometry of a mutant (alpha D60N) tryptophan synthase alpha 2 beta 2 complex reveals allosteric roles of alpha Asp60.

We have investigated the role of Asp60 of the alpha-subunit in allosteric communication between the tryptophan synthase alpha- and beta-subunits. Crystallographic and microspectrophotometric studies have been carried out on a mutant (alpha D60N) tryptophan synthase alpha 2 beta 2 complex which has no observable alpha-activity, but has substantial beta-activity. Single-crystal polarized absorption spectra indicate that the external aldimine is the predominant L-serine intermediate and that the amount of the intermediate formed is independent of pH, monovalent cations, and allosteric effectors. The three-dimensional structure is reported for this mutant enzyme complexed with indole 3-propanol phosphate bound to the alpha-site and L-serine bound to the beta-site (alpha D60N-IPP-Ser), and this structure is compared with that of the unliganded mutant enzyme (alpha D60N). In the complex, L-serine forms a stable external aldimine with the pyridoxal phosphate coenzyme at the active site of the beta-subunit. The conformation of the unliganded mutant is almost identical to that of the wild type enzyme. However, the structure of the mutant complexed with IPP and serine exhibits ligand-induced conformational changes much smaller than those observed previously for another mutant enzyme in the presence of the same ligands (beta K87T-IPP-Ser) [Rhee, S., Parris, K. D., Hyde, C. C., Ahmed, S. A., Miles, E. W., and Davies, D. R. (1997) Biochemistry 36, 7664-7680]. The alpha D60N-IPP-Ser alpha 2 beta 2 complex does not undergo the following ligand-induced conformational changes: (1) the closure of the alpha-subunit loop 6 (residues 178-191), (2) the movement of the mobile subdomain (residues 93-189) of the beta-subunit, and (3) the rotation of the alpha-subunit relative to the beta-subunit. These observations show that alpha Asp60 plays important roles in the closure of loop 6 and in allosteric communication between the alpha- and beta-subunits.

Allosteric Regulation↗

PubMed1998

Catalytic competence of O-acetylserine sulfhydrylase in the crystal probed by polarized absorption microspectrophotometry.

The reactions of the pyridoxal 5'-phosphate-dependent enzyme O-acetylserine sulfhydrylase with the substrate O-acetyl-L-serine and substrate analogs have been investigated in the crystalline state by single-crystal polarized absorption microspectrophotometry. This approach has allowed us to examine the catalytic competence of the enzyme in different crystalline states, one of which was used to determine the three-dimensional structure; experimental conditions were defined for the accumulation of catalytic intermediates in the crystal suitable for crystallographic analyses.O-Acetyl-L-serine reacts with the enzyme in one of the crystal forms leading via a beta-elimination reaction to the accumulation of the alpha-aminoacrylate Schiff base, absorbing maximally at 320 and 470 nm, as in solution. The dissociation constant for the alpha-aminoacrylate Schiff base is in the millimolar range, 500-fold higher than in solution, suggesting that crystal lattice interactions may oppose functionally relevant conformational changes. The dissociation constant exhibits a bell-shaped dependence on pH centered at pH 7. At this pH the alpha-aminoacrylate species slowly decays with time (30% decrease in 24 hours). The alpha-aminoacrylate intermediate readily reacts with sodium azide, an analog of sulfide, the natural nucleophilic agent, to give a new amino acid and the native enzyme, indicating that the crystalline enzyme catalyzes the overall beta-replacement reaction as in solution. In other crystal forms, including that used for the X-ray investigation, O-acetyl-L-serine either has an even higher dissociation constant or causes crystal damage upon binding. When the crystalline enzyme reacts with either L-cysteine or L-serine, the external aldimine intermediate is formed. The dissociation constants for both substrate analogs are closer to those observed in solution and are modulated by pH as in solution. Findings demonstrate that O-acetylserine sulfhydrylase is catalytically competent in the crystal although some regions of the molecule, likely involved in an open-closed transition induced by O-acetyl-L-serine binding, may have a limited flexibility. The accumulation in the crystal of both the external aldimine and the alpha-aminoacrylate intermediate makes feasible their structural determination and, therefore, the elucidation of the catalytic pathway at the molecular level.

Alanine↗

PubMed1997

Structure and oxygen affinity of crystalline des-his-146beta human hemoglobin in the T state.

To correlate directly structure with function, the oxygen affinity and the three-dimensional structure of crystals of the T quaternary state of des-His-146beta human hemoglobin have been determined by polarized absorption microspectrophotometry and x-ray diffraction crystallography. In des-His-146beta, the COOH-terminal histidine residues of the beta chains of hemoglobin A have been removed. Oxygen binding to crystalline des-His hemoglobin is non-cooperative and independent of pH. The oxygen affinity is 1.7-fold greater than that of the crystalline state of hemoglobin A. Removal of His-146beta results in a small movement of the truncated COOH-terminal peptide and in a very small change in quaternary structure. Previously, similar studies on T state crystals of des-Arg-141alpha hemoglobin showed that removal of the COOH termini of the alpha chains results in much larger effects on oxygen affinity and on quaternary structure. Kinetic studies in solution reveal that at pH 7.0, the rates of CO combination with deoxygenated des-His-146beta in the absence and presence of inositol hexaphosphate are 2.5- and 1.3-fold, respectively, more rapid than for hemoglobin A. The values for des-Arg are 7.6- and 3.9-fold. The properties of the T state of hemoglobin both in the crystal and in solution are influenced to a greater degree by the interactions associated with Arg-141alpha than those associated with His-146beta.

Computer Simulation↗

PubMed1997

T state hemoglobin binds oxygen noncooperatively with allosteric effects of protons, inositol hexaphosphate, and chloride.

Hemoglobin is the paradigm of allosteric proteins. Over the years, cooperative oxygen binding has been explained by different models predicting that the T state of hemoglobin binds oxygen either noncooperatively or with some degree of cooperativity or with strong cooperativity. Therefore, a critical test that discriminates among models is to determine the oxygen binding by the T state of hemoglobin. Fixation of hemoglobin in the T state has been achieved either by crystallization from polyethylene glycol solutions or by encapsulation in wet porous silica gels. Hemoglobin crystals bind oxygen noncooperatively with reduced affinity compared with solution, with no Bohr effect and with no influence of other allosteric effectors. In this study, we have determined accurate oxygen-binding curves to the T state of hemoglobin in silica gels with the same microspectrophotometric apparatus and multiwavelengths analysis used in crystal experiments. The T state of hemoglobin in silica gels binds oxygen noncooperatively with an affinity and a Bohr effect similar to those observed in solution for the binding of the first oxygen molecule. Other allosteric effectors such as inositol hexaphosphate, bezafibrate, and chloride significantly affect oxygen affinity. Therefore, T state hemoglobins that are characterized by strikingly different functional properties share the absence of cooperativity in the binding of oxygen. These findings are fully consistent with the Monod, Wyman, and Changeux model and with most features of Perutz's stereochemical model, but they are not consistent with models of both Koshland and Ackers.

Allosteric Regulation↗

PubMed1997

Time-resolved fluorescence of O-acetylserine sulfhydrylase catalytic intermediates.

The reaction of the substrate O-acetyl-L-serine (OAS) with the pyridoxal 5'-phosphate (PLP)-dependent enzyme O-acetylserine sulfhydrylase-A (OASS-A) proceeds via the transient formation of an external aldimine absorbing at 420 nm and a stable alpha-aminoacrylate intermediate absorbing at 330 and 465 nm. Stable external aldimine species are obtained by reaction of the enzyme with either the reaction product L-cysteine or the product analog L-serine. Static and time-resolved fluorescence emission properties of the coenzyme in the above catalytic intermediates have been used to directly probe the active site conformation at different stages of the catalytic pathway. Upon excitation at either 420 or 330 nm, the external aldimines with L-cysteine and L-serine exhibit a structured emission centered at 490 nm with a shoulder at 530 nm. Fluorescence decays upon excitation at 420 nm are best fitted using two components with lifetimes of 1.1 and 3.8 ns, with the fractional intensity of the slow component being 0.92 with L-cysteine and 0.75 with L-serine, respectively. The fast component, emitting at 530 nm, is attributed to a dipolar species formed in the excited state by proton dissociation, and the slow component, emitting at 490 nm, is attributed to a ketoenamine tautomer of the external aldimine. The slow component for external aldimine fluorescence decay is characterized by the same lifetime value as that of the internal aldimine with an increased fractional intensity, indicating that the distribution between the ketoenamine and the dipolar species is shifted toward the ketoenamine tautomer in the external aldimine, compared to the internal aldimine. Differences in equilibrium distribution of ketoenamine and enolimine tautomers can also account for differences in the emission properties of the external aldimines of L-cysteine and L-serine. The alpha-aminoacrylate species is characterized by a relatively weak emission. Upon excitation at 330 nm, the emission exhibits two bands centered at 420 and 540 nm, whereas upon excitation at 420 nm the emission bands are centered at 500 and 540 nm, and upon excitation at 465 nm, the main absorbance peak of the alpha-aminoacrylate species, the emission spectrum shows a band at 540 nm. The fluorescence decays, upon excitation at 330 nm, are best fitted using three components with lifetime values similar to those found for the internal aldimine, with the slow component predominating. Species-associated spectra, collected between 400 and 520 nm upon excitation at 350 nm, indicate the presence of a fast component overlapping the slow component on the blue side of the emission spectrum, as detected for the internal aldimine. When the excitation wavelength is 420 nm, there are only two components with the fast one predominating. A further increase in the fractional intensity of the fast component is observed upon excitation at 465 nm. The weak emission and the short lifetime of the emission excited at 465 nm indicate that this alpha-aminoacrylate tautomer interacts significantly with neighboring groups of the protein matrix and may be endowed with a higher mobility than the external aldimine.

Catalysis↗

PubMed1997

Crystal structures and inhibitor binding in the octameric flavoenzyme vanillyl-alcohol oxidase: the shape of the active-site cavity controls substrate specificity.

BACKGROUND: Lignin degradation leads to the formation of a broad spectrum of aromatic molecules that can be used by various fungal micro-organisms as their sole source of carbon. When grown on phenolic compounds, Penicillium simplicissimum induces the strong impression of a flavin-containing vanillyl-alcohol oxidase (VAO). The enzyme catalyses the oxidation of a vast array of substrates, ranging from aromatic amines to 4-alkyphenols. VAO is a member of a novel class of widely distributed oxidoreductases, which use flavin adenine dinucleotide (FAD) as a cofactor covalently bound to the protein. We have carried out the determination of the structure of VAO in order to shed light on the most interesting features of these novel oxidoreductases, such as the functional significance of covalent flavinylation and the mechanism of catalysis. RESULTS: The crystal structure of VAO has been determined in the native state and in complexes with four inhibitors. The enzyme is an octamer with 42 symmetry; the inhibitors bind in a hydrophobic, elongated cavity on the si side of the flavin molecule. Three residues, Tyr108, Tyr503 and Arg504 form an anion-binding subsite, which stabilises the phenolate form of the substrate. The structure of VAO complexed with the inhibitor 4-(1-heptenyl)phenol shows that the catalytic cavity is completely filled by the inhibitor, explaining why alkylphenols bearing aliphatic substituents longer than seven carbon atoms do not bind to the enzyme. CONCLUSIONS: The shape of the active-site cavity controls substrate specificity by providing a 'size exclusion mechanism'. Inside the cavity, the substrate aromatic ring is positioned at an angle of 18 degrees to the flavin ring. This arrangement is ideally suited for a hydride transfer reaction, which is further facilitated by substrate deprotonation. Burying the substrate beneath the protein surface is a recurrent strategy, common to many flavoenzymes that effect substrate oxidation or reduction via hydride transfer.

Alcohol Oxidoreductases↗

PubMed1997

Active site plasticity in D-amino acid oxidase: a crystallographic analysis.

D-Amino acid oxidase (DAAO) is the prototype of the flavin-containing oxidases. It catalyzes the oxidative deamination of various D-amino acids, ranging from D-Ala to D-Trp. We have carried out the X-ray analysis of reduced DAAO in complex with the reaction product imino tryptophan (iTrp) and of the covalent adduct generated by the photoinduced reaction of the flavin with 3-methyl-2-oxobutyric acid (kVal). These structures were solved by combination of 8-fold density averaging and least-squares refinement techniques. The FAD redox state of DAAO crystals was assessed by single-crystal polarized absorption microspectrophotometry. iTrp binds to the reduced enzyme with the N, C alpha, C, and C beta atoms positioned 3.8 A from the re side of the flavin. The indole side chain points away from the cofactor and is bound in the active site through a rotation of Tyr224. This residue plays a crucial role in that it adapts its conformation to the size of the active site ligand, providing the enzyme with the plasticity required for binding a broad range of substrates. The iTrp binding mode is fully consistent with the proposal, inferred from the analysis of the native DAAO structure, that substrate oxidation occurs via direct hydride transfer from the C alpha to the flavin N5 atom. In this regard, it is remarkable that, even in the presence of the bulky iTrp ligand, the active center is made solvent inaccessible by loop 216-228. This loop is thought to switch between the "closed" conformation observed in the crystal structures and an "open" state required for substrate binding and product release. Loop closure is likely to have a role in catalysis by increasing the hydrophobicity of the active site, thus making the hydride transfer reaction more effective. Binding of kVal leads to keto acid decarboxylation and formation of a covalent bond between the keto acid C alpha and the flavin N5 atoms. Formation of this acyl adduct results in a nonplanar flavin, characterized by a 22 degrees angle between the pyrimidine and benzene rings. Thus, in addition to an adaptable substrate binding site, DAAO has the ability to bind a highly distorted cofactor. This ability is relevant for the enzyme's function as a highly efficient oxidase.

Arginine↗

PubMed1997

Allosteric effectors do not alter the oxygen affinity of hemoglobin crystals.

In solution, the oxygen affinity of hemoglobin in the T quaternary structure is decreased in the presence of allosteric effectors such as protons and organic phosphates. To explain these effects, as well as the absence of the Bohr effect and the lower oxygen affinity of T-state hemoglobin in the crystal compared to solution, Rivetti C et al. (1993a, Biochemistry 32:2888-2906) suggested that there are high- and low-affinity subunit conformations of T, associated with broken and unbroken intersubunit salt bridges. In this model, the crystal of T-state hemoglobin has the lowest possible oxygen affinity because the salt bridges remain intact upon oxygenation. Binding of allosteric effectors in the crystal should therefore not influence the oxygen affinity. To test this hypothesis, we used polarized absorption spectroscopy to measure oxygen binding curves of single crystals of hemoglobin in the T quaternary structure in the presence of the "strong" allosteric effectors, inositol hexaphosphate and bezafibrate. In solution, these effectors reduce the oxygen affinity of the T state by 10-30-fold. We find no change in affinity (< 10%) of the crystal. The crystal binding curve, moreover, is noncooperative, which is consistent with the essential feature of the two-state allosteric model of Monod J, Wyman J, and Changeux JP (1965, J Mol Biol 12:88-118) that cooperative binding requires a change in quaternary structure. Noncooperative binding by the crystal is not caused by cooperative interactions being masked by fortuitous compensation from a difference in the affinity of the alpha and beta subunits. This was shown by calculating the separate alpha and beta subunit binding curves from the two sets of polarized optical spectra using geometric factors from the X-ray structures of deoxygenated and fully oxygenated T-state molecules determined by Paoli M et al. (1996, J Mol Biol 256:775-792).

Allosteric Regulation↗

PubMed1996

Time-resolved fluorescence of tryptophan synthase.

Time-resolved and steady-state fluorescence of the tryptophan synthase alpha 2 beta 2 complex and of the beta 2 dimer from Salmonella typhimurium were measured to characterize the conformational properties of the beta subunit in the presence and in the absence of the alpha subunit when the catalytic species internal aldimine, external aldimine and alpha-aminoacrylate Schiff bases were selectively accumulated within the beta active site. The fluorescence decay of the coenzyme pyridoxal 5'-phosphate, bound via a Schiff base in the beta subunit of the alpha 2 beta 2 complex (internal aldimine species), is accounted for by two lifetimes (2.9 and 0.9 ns) of almost equal fractional intensity that are slightly affected by pH. Accordingly, both the absorption and emission spectra were found to be pH independent. The emission properties of the internal aldimine in the beta 2 dimer are pH dependent, suggesting that the alpha-subunit binding alters the microenvironment of the beta-subunit active site. This conclusion is also supported by the emission of the single tryptophanyl residue of the enzyme (Trp-177 beta). In the reaction of L-serine with the alpha 2 beta 2 complex, the predominant catalytic intermediate is the external aldimine (lambda(max) = 422 nm) at pH 10, and the alpha-aminoacrylate (lambda(max) = 350 nm) at pH 7. The external aldimine exhibits a high fluorescence intensity at 500 nm that decays with a single lifetime of 6.2 ns in the alpha 2 beta 2 complex, at pH 10, and at a similar value in the beta 2 dimer. The emission properties of the external aldimine with respect to the internal aldimine, and the small effects induced by alpha-subunit binding indicate a shielding of the coenzyme and a stabilization of its excited state. In contrast, the short fluorescence lifetime (0.4 ns) and the weak fluorescence emission of the alpha-aminoacrylate Schiff base indicate an increase of non-radiative processes possibly due to a more tight coupling of this intermediate with the protein matrix with respect to the external aldimine. Whereas the internal aldimine is distributed in two tautomeric forms, both the external aldimine and the alpha-aminoacrylate are present in single conformational states with distinct structural and/or dynamic properties that may modulate regulatory intersubunit signals.

Bacterial Proteins↗

PubMed1996

Cooperative oxygen binding to scapharca inaequivalvis hemoglobin in the crystal.

Oxygen binding to homodimeric Scapharca inaequivalvis hemoglobin (HbI) crystals has been investigated by single-crystal polarized absorption microspectrophotometry. The saturation curve, characterized by a Hill coefficient nH = 1.45 and an oxygen pressure at half saturation p50 = 4.8 torr, at 15 degrees C, shows that HbI in the crystalline state retains positive cooperativity in ligand binding. This finding will permit the correlation of the oxygen-linked conformational changes in the crystal with the expression of cooperativity. Polarized absorption spectra of deoxy-HbI, oxy-HbI, and oxidized HbI crystals indicate that oxygenation does not induce heme reorientation, whereas oxidation does. Lattice interactions prevent the dissociation of oxidized dimers that occurs in solution and stabilize an equilibrium distribution of pentacoordinate and hexacoordinate high spin species.

Animals↗

PubMed1996

Allosteric regulation of tryptophan synthase: effects of pH, temperature, and alpha-subunit ligands on the equilibrium distribution of pyridoxal 5'-phosphate-L-serine intermediates.

The equilibrium distribution of catalytic intermediates formed in the reaction of L-serine with the tryptophan synthase alpha 2 beta 2-complex from Salmonella typhimurium has been investigated by absorption and fluorescence spectroscopy as a function of pH, temperature, and alpha-subunit ligands. The novel result of this study is that the equilibrium between the two major catalytic species, the external aldimine and the alpha-aminoacrylate, is modulated by the ionization of two groups with apparent pK values of 7.8 +/- 0.3 and 10.3 +/- 0.2. Protonation of these groups stabilizes the alpha-aminoacrylate Schiff base by an estimated 100-fold with respect to the external aldimine. Furthermore, the formation of the alpha-aminoacrylate from the external aldimine is an endothermic process. Temperature slightly affects the apparent pK values but remarkably influences the amplitude of the phase associated with the ionization of each group. At 20 degrees C, each phase accounts for nearly half of the titration. Since the isolated beta 2-dimer does not exhibit a pH-dependent distribution of intermediates, the alpha-beta-subunit interactions seem critical to the onset of this functional property of the beta-subunit. The modulation of intersubunit interactions by the alpha-subunit ligands DL-alpha-glycerol 3-phosphate and phosphate leads to significant changes in the pH-dependent distribution of intermediates. At saturating concentrations of either of these alpha-subunit ligands, the alpha-aminoacrylate Schiff base is the predominant species over a wide pH range while the apparent pK values of the groups that control the equilibrium are not significantly affected. The pH-dependent interconversion of catalytic intermediates here reported has not been previously detected because phosphate buffers have usually been employed in the studies of this enzyme. Our findings are discussed in the light of a model in which specific protein conformations are associated with the external aldimine and the alpha-aminoacrylate Schiff bases, the latter being stabilized by temperature, protons, and alpha-subunit ligands.

Allosteric Regulation↗

PubMed1996

Oxygen binding by single crystals of hemoglobin: the problem of cooperativity and inequivalence of alpha and beta subunits.

Oxygen binding by the human hemoglobin tetramer in the T quaternary structure is apparently noncooperative in the crystalline state (Hill n = 1.0), as predicted by the two-state allosteric model of Monod, Wyman, and Changeux (MWC) (Mozzarelli et al., Nature 351:416-419, 1991; Rivetti et al., Biochemistry 32:2888-2906, 1993). However, cooperativity within the tetramer can be masked by a difference in affinity between the alpha and beta subunits. Indeed, analysis of the binding curves derived from absorption of light polarized along two different crystal directions, for which the projections of the alpha and beta hemes are slightly different, revealed an inequivalence in the intrinsic oxygen affinity of the alpha and beta subunits (p50(alpha) approximately 80 torr, p50(beta) approximately 370 torr at 15 degrees C) that compensates a small amount of cooperativity (Rivetti et al., Biochemistry 32:2888-2906, 1993). To further investigate this problem, we have measured oxygen binding curves of single crystals of hemoglobin (in a different lattice) in which the iron in the alpha subunits has been replaced by the non-oxygen-binding nickel(II). The Hill n is 0.90 +/- 0.06, and the p50 is slightly different for light polarized parallel to different crystal directions, indicating a very small difference in affinity between the two crystallographically inequivalent beta subunits. The average crystal p50 is 110 +/- 20 torr at 15 degrees C, close to the p50 of 80 torr observed in solution, but about threefold less than the p50 calculated by Rivetti et al. (Biochemistry 32:2888-2906, 1993) for the beta subunits of the unsubstituted tetramer. These results suggest that Rivetti et al., if anything, overestimated the alpha/beta inequivalence. They therefore did not underestimate the cooperativity within the T quaternary structure, when they concluded that it represents a small deviation from the perfectly noncooperative binding of an MWC allosteric model. Our conclusion of nearly perfect MWC behavior for binding to the T state of unmodified hemoglobin raises the question of the relevance of the large T-state cooperativity inferred for cyanide binding to partially oxidized hemoglobin (Ackers et al., Science 255:54-63, 1992).

Allosteric Site↗

PubMed1996

Protein function in the crystal.

Protein crystals contain wide solvent-filled channels that allow for traffic of metabolites and intramolecular motility. Ligand binding, catalysis and allosteric regulation occur in the crystalline environment but intermolecular interactions may hinder function-associated transitions and alter activity with respect to solution. Lattice constraints have, however, provided the opportunity to isolate and characterize conformational states that are poorly populated in solution. New methods are being developed to initiate reactions rapidly and synchronously throughout the crystal and to monitor their time course. A model consistent with kinetics in the crystal is necessary to interpret the results of time-resolved macromolecular crystallography.

Allosteric Regulation↗

PubMed1995

Monovalent cations affect dynamic and functional properties of the tryptophan synthase alpha 2 beta 2 complex.

Monovalent cations affect both conformational and catalytic properties of the tryptophan synthase alpha 2 beta 2 complex from Salmonella typhimurium. Their influence on the dynamic properties of the enzyme was probed by monitoring the phosphorescence decay of the unique Trp-177 beta, a residue located near the beta-active site, at the interface between alpha- and beta-subunits. In the presence of either Li+, Na+, Cs+, or NH4+, the phosphorescence decay is biphasic and the average lifetime increases indicating a decrease in the flexibility of the N-terminal domain of the beta-subunit. Since amplitudes but not lifetimes are affected, cations appear to shift the equilibrium between preexisting enzyme conformations. The effect on the reaction between indole and L-serine was studied by steady state kinetic methods at room temperature. We found that cations: (i) bind to the L-serine--enzyme derivatives with an apparent dissociation constant, measured as the concentration of cation corresponding to one-half of the maximal activity, that is in the millimolar range and decreases with ion size; (ii) increase kcat with the order of efficacy Cs+ > K+ > Li+ > Na+; (iii) decrease KM for indole, Na+ being the most effective and causing a 30-fold decrease; and (iv) cause an increase of the kcat/KM ratio by 20-40-fold. The influence on the equilibrium distribution between the external aldimine and the alpha-aminoacrylate, intermediates in the reaction of L-serine with the beta-subunits of the enzyme, was found to be cation-specific.(ABSTRACT TRUNCATED AT 250 WORDS)

Ammonia↗

PubMed1995

Structure and oxygen affinity of crystalline desArg141 alpha human hemoglobin A in the T state.

The correlation of a protein structure determined crystallographically to its functional properties determined in solution can be an extremely complex problem due to potential differences of protein conformational flexibility in the two physical states. A more direct approach to the correlation of structure with function is to examine both the structure and the function of a protein in the same crystalline environment. In this paper, the structural and functional properties of T state desArg hemoglobin (human hemoglobin modified by removal of the alpha-chain COOH-terminal residue, Arg141 alpha) have been studied in the same crystal form by high resolution X-ray diffraction methods and by polarized absorption microspectrophotometry. Specifically, the crystal structure of deoxygenated desArg human hemoglobin has been refined at a 2.1 A resolution using crystals grown at low salt concentration from solutions of polyethylene glycol. The loss of Arg141 alpha and all of the salt bridges in which it participates is associated with subtle structural perturbations of the alpha-chains which include an increase in the conformational flexibility of both the NH2 and COOH-terminal peptides. Although the heme pockets appear unchanged and even the side-chain of Tyr140 is oriented nearly as in HbA, the functional characterization by microspectrophotometric measurements indicates that crystals of desArg hemoglobin bind oxygen with an affinity which is roughly 15-fold greater than that of crystals of human hemoglobin A. There is no alkaline Bohr effect or effect of chloride ions, but an acid Bohr effect is observed. The oxygen affinities measured along two principal axes of the crystals differ by 25%, indicating heterogeneity in the affinities of the oxygen binding sites. This finding and the measured Hill coefficient of unity suggest significant cooperativity in the binding of oxygen in these crystals. The origins of the observed heterogeneity and the implied cooperativity are unknown.

Arginine↗

PubMed1993

Effect of chloride on oxygen binding to crystals of hemoglobin Rothschild (beta 37 Trp-->Arg) in the T quaternary structure.

Oxygen binding to crystals of hemoglobin Rothschild (beta 37 Trp-->Arg) in the T quaternary structure has been investigated by polarized absorption microspectrophotometry. These crystals were grown from poly(ethylene glycol) solutions containing low concentrations of salt. In the absence of chloride, they have a significantly higher oxygen affinity than crystals of human hemoglobin A grown in a similar manner, and exhibit Hill coefficients lower than 1. There is no Bohr effect from pH 6 to 9. We have found that chloride decreases the oxygen affinity of Hb Rothschild crystals, an effect which is absent in crystals of HbA. This dependence of affinity on chloride is almost certainly associated with the chloride binding sites which have been localized crystallographically at the mutant arginine residues (Kavanaugh et al., 1992). Since chloride binding appears to lower the oxygen affinities of both the alpha and beta chains, the linkage between the binding of oxygen and the dissociation of chloride results in significant cooperativity in oxygen binding to the crystals.

Amino Acid Sequence↗