Stefano Bettati

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.

Evolution of allosteric models for hemoglobin

We compare various allosteric models that have been proposed to explain cooperative oxygen binding to hemoglobin, including the two‐state allosteric model of Monod, Wyman, and Changeux (MWC), the Cooperon model of Brunori, the model of Szabo and Karplus (SK) based on the stereochemical mechanism of Perutz, the generalization of the SK model by Lee and Karplus (SKL), and the Tertiary Two‐State (TTS) model of Henry, Bettati, Hofrichter and Eaton. The preponderance of experimental evidence favors the TTS model which postulates an equilibrium between high (r)‐ and low (t)‐affinity tertiary conformations that are present in both the T and R quaternary structures. Cooperative oxygenation in this model arises from the shift of T to R, as in MWC, but with a significant population of both r and t conformations in the liganded T and in the unliganded R quaternary structures. The TTS model may be considered a combination of the SK and SKL models, and these models provide a framework for a structural interpretation of the TTS parameters. The most compelling evidence in favor of the TTS model is the nanosecond ‐ millisecond carbon monoxide (CO) rebinding kinetics in photodissociation experiments on hemoglobin encapsulated in silica gels. The polymeric network of the gel prevents any tertiary or quaternary conformational changes on the sub‐second time scale, thereby permitting the subunit conformations prior to CO photodissociation to be determined from their ligand rebinding kinetics. These experiments show that a large fraction of liganded subunits in the T quaternary structure have the same functional conformation as liganded subunits in the R quaternary structure, an experimental finding inconsistent with the MWC, Cooperon, SK, and SKL models, but readily explained by the TTS model as rebinding to r subunits in T. We propose an additional experiment to test another key prediction of the TTS model, namely that a fraction of subunits in the unliganded R quaternary structure has the same functional conformation (t) as unliganded subunits in the T quaternary structure.

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°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 α‐ and β‐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.

Interaction of serine acetyltransferase with O-acetylserine sulfhydrylase active site: Evidence from fluorescence spectroscopy

Serine acetyltransferase is a key enzyme in the sulfur assimilation pathway of bacteria and plants, and is known to form a bienzyme complex with O‐acetylserine sulfhydrylase, the last enzyme in the cysteine biosynthetic pathway. The biological function of the complex and the mechanism of reciprocal regulation of the constituent enzymes are still poorly understood. In this work the effect of complex formation on the O‐acetylserine sulfhydrylase active site has been investigated exploiting the fluorescence properties of pyridoxal 5′‐phosphate, which are sensitive to the cofactor microenvironment and to conformational changes within the protein matrix. The results indicate that both serine acetyltransferase and its C‐terminal decapeptide bind to the α‐carboxyl subsite of O‐acetylserine sulfhydrylase, triggering a transition from an open to a closed conformation. This finding suggests that serine acetyltransferase can inhibit O‐acetylserine sulfhydrylase catalytic activity with a double mechanism, the competition with O‐acetylserine for binding to the enzyme active site and the stabilization of a closed conformation that is less accessible to the natural substrate.

Unfolding of Green Fluorescent Protein mut2 in wet nanoporous silica gels

Many of the effects exerted on protein structure, stability, and dynamics by molecular crowding and confinement in the cellular environment can be mimicked by encapsulation in polymeric matrices. We have compared the stability and unfolding kinetics of a highly fluorescent mutant of Green Fluorescent Protein, GFPmut2, in solution and in wet, nanoporous silica gels. In the absence of denaturant, encapsulation does not induce any observable change in the circular dichroism and fluorescence emission spectra of GFPmut2. In solution, the unfolding induced by guanidinium chloride is well described by a thermodynamic and kinetic two‐state process. In the gel, biphasic unfolding kinetics reveal that at least two alternative conformations of the native protein are significantly populated. The relative rates for the unfolding of each conformer differ by almost two orders of magnitude. The slower rate, once extrapolated to native solvent conditions, superimposes to that of the single unfolding phase observed in solution. Differences in the dependence on denaturant concentration are consistent with restrictions opposed by the gel to possibly expanded transition states and to the conformational entropy of the denatured ensemble. The observed behavior highlights the significance of investigating protein function and stability in different environments to uncover structural and dynamic properties that can escape detection in dilute solution, but might be relevant for proteins in vivo.

Design of O-acetylserine sulfhydrylase inhibitors by mimicking Nature

The inhibition of cysteine biosynthesis in prokaryotes and protozoa has been proposed to be relevant for the development of antibiotics. Haemophilus influenzae O-acetylserine sulfhydrylase (OASS), catalyzing l-cysteine formation, is inhibited by the insertion of the C-terminal pentapeptide (MNLNI) of serine acetyltransferase into the active site. Four-hundred MNXXI pentapeptides were generated in silico, docked into OASS active site using GOLD, and scored with HINT. The terminal P5 Ile accounts for about 50% of the binding energy. Glu or Asp at position P4 and, to a lesser extent, at position P3 also significantly contribute to the binding interaction. The predicted affinity of 14 selected pentapeptides correlated well with the experimentally determined dissociation constants. The X-ray structure of three high affinity pentapeptide-OASS complexes were compared with the docked poses. These results, combined with a GRID analysis of the active site, allowed us to define a pharmacophoric scaffold for the design of peptidomimetic inhibitors.

Lung metastasis resection of adenoid cystic carcinoma of salivary glands

BACKGROUND Adenoid cystic carcinoma is a rare tumour originating from the exocrine mucous glands, known for its high propensity for distant metastases. The value of lung metastasis resection from adenoid cystic carcinoma of salivary glands origin is evaluated. METHODS A retrospective study was conducted on patients undergoing surgery for primary adenoid cystic carcinoma of the salivary glands between 1982 and 2006. Patients were excluded who had primary tumour macroscopic incomplete resection or were lost at follow-up. From a database of 50 eligible patients, 27 were identified as having presented a tumour recurrence during follow-up; in 20 it was first diagnosed in the form of distant metastases, and in 7 in the form of loco-regional recurrence. Nine patients who presented isolated lung recurrence underwent complete lung metastasectomy. Demographic data, pathologic characteristics and operative and postoperative record were reviewed, as well as updated survival. RESULTS Twenty-six men and 24 women with a median age of 57 years (range 33-79) underwent radical surgery for adenoid cystic carcinoma during the study period. In 20 patients, at a median free interval time of 3 years (range 1-12), a distant metastasis relapse was observed. Nine patients with a median free interval time of 5 years (range 1-12) underwent lung metastasectomy: five had single metastasis resection, one multiple mono-pulmonary and three multiple and bilateral. In six of these patients a new disease recurrence was noted: four patients underwent further lung metastasectomy, but in all of them progression of the disease was observed. Mean survival of the population as a whole resulted as being 16 years (SE=1.4) with an actuarial survival of 77% at 5 years, 66% at 10 years and 56% at 15 years. Mean survival of patients having presented with distant metastases resulted as being 11 years (SE=2.2). Mean survival after appearance of distant metastases resulted as being 72 months (SE=15.8) in the 9 patients treated by metastasectomy, and 62 months (SE=15.1) in the 11 who did not have metastasis resection. CONCLUSIONS Patients with adenoid cystic carcinoma could be frequently encountered with disease recurrence confined to the lung. The impact of complete lung metastasis resection on the course of the disease, however, is yet to be determined.

A Two-step Process Controls the Formation of the Bienzyme Cysteine Synthase Complex

The regulation of enzyme activity through the transient formation of multiprotein assemblies plays an important role in the control of biosynthetic pathways. One of the first regulatory complexes to be discovered was cysteine synthase (CS), formed by the pyridoxal 5′-phosphate-dependent enzyme O-acetylserine sulfhydrylase (OASS) and serine acetyltransferase (SAT). These enzymes are at the branch point of the sulfur, carbon, and nitrogen assimilation pathways. Understanding the mechanism of complex formation helps to clarify the role played by CS in the regulation of sulfur assimilation in bacteria and plants. To this goal, stopped-flow fluorescence spectroscopy was used to characterize the interaction of SAT with OASS, at different temperatures and pH values, and in the presence of the physiological regulators cysteine and bisulfide. Results shed light on the mechanism of complex formation and regulation, so far poorly understood. Cysteine synthase assembly occurs via a two-step mechanism involving rapid formation of an encounter complex between the two enzymes, followed by a slow conformational change. The conformational change likely results from the closure of the active site of OASS upon binding of the SAT C-terminal peptide. Bisulfide, the second substrate and a feedback inhibitor of OASS, stabilizes the CS complex mainly by decreasing the back rate of the isomerization step. Cysteine, the product of the OASS reaction and a SAT inhibitor, slightly affects the kinetics of CS formation leading to destabilization of the complex.

Tyrosine phenol-lyase and tryptophan indole-lyase encapsulated in wet nanoporous silica gels: Selective stabilization of tertiary conformations.

The pyridoxal 5′‐phosphate‐dependent enzymes tyrosine phenol‐lyase and tryptophan indole‐lyase were encapsulated in wet nanoporous silica gels, a powerful method to selectively stabilize tertiary and quaternary protein conformations and to develop bioreactors and biosensors. A comparison of the enzyme reactivity in silica gels and in solution was carried out by determining equilibrium and kinetic parameters, exploiting the distinct spectral properties of catalytic intermediates and reaction products. The encapsulated enzymes exhibit altered distributions of ketoenamine and enolimine tautomers, increased values of inhibitors dissociation constants, slow attaining of steady‐state in the presence of substrate and substrate analogs, modified steady‐state distribution of catalytic intermediates, and a sixfold–eightfold decrease of specific activities. This behavior can be rationalized by a reduced conformational flexibility for the encapsulated enzymes and a selective stabilization of either the open (inactive) or the closed (active) form of the enzymes. Despite very similar structures and catalytic mechanisms, the influence of encapsulation is more pronounced for tyrosine phenol‐lyase than tryptophan indole‐lyase. This finding indicates that subtle structural and dynamic differences can lead to distinct interactions of the protein with the gel matrix.

The multifaceted pyridoxal 5'-phosphate-dependent O-acetylserine sulfhydrylase.

Cysteine is the final product of the reductive sulfate assimilation pathway in bacteria and plants and serves as the precursor for all sulfur-containing biological compounds, such as methionine, S-adenosyl methionine, iron-sulfur clusters and glutathione. Moreover, in several microorganisms cysteine plays a role as a reducing agent, eventually counteracting host oxidative defense strategies. Cysteine is synthesized by the PLP-dependent O-acetylserine sulfhydrylase, a dimeric enzyme belonging to the fold type II, catalyzing a beta-replacement reaction. In this review, the spectroscopic properties, catalytic mechanism, three-dimensional structure, conformational changes accompanying catalysis, determinants of enzyme stability, role of selected amino acids in catalysis, and the regulation of enzyme activity by ligands and interaction with serine acetyltransferase, the preceding enzyme in the biosynthetic pathway, are described. Given the key biological role played by O-acetylserine sulfhydrylase in bacteria, inhibitors with potential antibiotic activity have been developed. This article is part of a Special Issue entitled: Pyridoxal Phospate Enzymology.