1,721,043 research outputs found
Editorial: Chemically modified proteins and oligopeptides: A toolbox for therapeutics, diagnostics, and analytics
Full text linkKinetic characterization of the human O-phosphoethanolamine phospho-lyase reveals unconventional features of this specialized pyridoxal phosphate-dependent lyase
No full textHuman O-phosphoethanolamine (PEA) phospho-lyase is a pyridoxal 5′-phosphate (PLP) dependent enzyme that catalyzes the degradation of PEA to acetaldehyde, phosphate and ammonia. Physiologically, the enzyme is involved in phospholipid metabolism and is expressed mainly in the brain, where its expression becomes dysregulated in the course of neuropsychiatric diseases. Mechanistically, PEA phospho-lyase shows a remarkable substrate selectivity, strongly discriminating against other amino compounds structurally similar to PEA. Herein, we studied the enzyme under steady-state and pre-steady-state conditions, analyzing its kinetic features and getting insights into the factors that contribute to its specificity. The pH dependence of the catalytic parameters and the pattern of inhibition by the product phosphate and by other anionic compounds suggest that the active site of PEA phospho-lyase is optimized to bind dianionic groups and that this is a prime determinant of the enzyme specificity towards PEA. Single- and multiple-wavelength stopped-flow studies show that upon reaction with PEA the main absorption band of PLP (λmax = 412 nm) rapidly blue-shifts to ~ 400 nm. Further experiments suggest that the newly formed and rather stable 400-nm species most probably represents a Michaelis (noncovalent) complex of PEA with the enzyme. Accumulation of such an early intermediate during turnover is unusual for PLP-dependent enzymes and appears counterproductive for absolute catalytic performance, but it can contribute to optimize substrate specificity. PEA phospho-lyase may hence represent a case of selectivity–efficiency tradeoff. In turn, the strict specificity of the enzyme seems important to prevent inactivation by other amines, structurally resembling PEA, that occur in the brain
Tertiary and quaternary effects in the allosteric regulation of animal hemoglobins
Full text linkIn the last decade, protein allostery has experienced a major resurgence, boosted by the extension of the concept to systems of increasing complexity and by its exploitation for the development of drugs. Expansion of the field into new directions has not diminished the key role of hemoglobin as a test molecule for theory and experimental validation of allosteric models. Indeed, the diffusion of hemoglobins in all kingdoms of life and the variety of functions and of quaternary assemblies based on a common tertiary fold indicate that this superfamily of proteins is ideally suited for investigating the physical and molecular basis of allostery and firmly maintains its role as a main player in the field. This review is an attempt to briefly recollect common and different strategies adopted by metazoan hemoglobins, from monomeric molecules to giant complexes, exploiting homotropic and heterotropic allostery to increase their functional dynamic range
Immobilization of proteins in Ormosil gels: Functional properties and applications
No full textOrganically modified silica (Ormosil) gels are three-dimensional matrices obtained by co-polymerizing tetraalkyl orthosilicates and organoalkoxysilanes through the sol-gel technology. Ormosil matrices are highly flexible with respect to macroscopic size and shape, as they can be prepared in the form of monoliths, layers, powders and nanoparticles, and to their physical-chemical properties, with special regard to hydrophobicity or specific functionalization of their alkyl and aryl groups. This makes Ormosils a very promising matrix, though not widely exploited so far, for the immobilization of enzymes and other biomolecules either by encapsulation within the gel matrix or by surface decoration of nanoparticles. Potential applications range from the development of biocatalysts and bioremediation devices to intracellular drug delivery and photodynamic therapy
Discovery of a new intermediate in R-T transition of human hemoglobin by silica gel encapsulation
No full textCO rebinding kinetics to myoglobin- and R state hemoglobin-doped silica gels in the presence of glycerol
No full textThis study characterizes the CO rebinding kinetics after photodissociation of horse heart myoglobin (Mb) and human R-state hemoglobin (Hb) encapsulated in wet silica gels, in the presence of various concentrations of glycerol. The geminate yield for HbCO is scarcely affected by the gel matrix, indicating that the protein can fluctuate as in a homogeneous solution. On the contrary, the geminate yield for gel-embedded MbCO is much higher than that in solution, suggesting that the gel matrix inhibits the movements of the protein. The geminate yield for both proteins increases substantially with the addition of glycerol to the bathing solution. The observed kinetics could be rationalized using a simple three-state model. Rate constants have been modeled using a modified Kramers equation, which indicated that the gel exerts an internal friction on the elementary rate constants. The rate constant for geminate rebinding, kCA, is essentially viscosity independent below 20 cP for both proteins. The internal friction for the ligand escape rate, kCS, is much smaller and is found to be negligible for HbCO and 5 cP for MbCO. The activation barrier for kCS increases with glycerol concentration in response to increased viscosity and reduced ligand solubility. The rate kSC showed a complex behavior that reflects the opposing effects of viscosity and activity arising from molecular confinement and crowding. Accordingly, the corresponding activation barriers show a biphasic behavior, with a minimum at ≈40% glycerol for HbCO and at ≈75% glycerol for MbCO. The results highlight the potential of silica gel encapsulation for in vitro studies aimed to reproduce the crowded and confined environment experienced by proteins in vivo. The diverse response to encapsulation of Mb and Hb could actually reflect physiologically relevant functional properties escaping detection in the diluted solutions normally used for biophysical investigations
Polymerized and polyethylene glycol-conjugated hemoglobins: A globin-based calibration curve for dynamic light scattering analysis.
No full textTrapping hemoglobin in rigid matrices: fine tuning of oxygen binding properties by modulation of encapsulation protocols
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