RESUMEN
Human carbonic anhydrase II catalyzes the reversible reaction of carbon dioxide and water to form bicarbonate and a proton. His64-mediated proton shuttling between the active site and the bulk solvent is rate limiting. Here we investigate the protonation behavior of His64 as well as its structural and dynamic features in a pH dependent way. We derive two pKa values for His64, 6.25 and 7.60, that we were able to assign to its inward and outward conformation. Furthermore, we show that His64 exists in both conformations equally, independent of pH. Both conformations display an equal distribution of their two neutral tautomeric states. The life time of each conformation is short and both states display high flexibility within their orientation. Therefore, His64 is never static, but rather poised to change conformation. These findings support an energetic, dynamic and solution ensemble-based framework for the high enzymatic activity of human carbonic anhydrase II.
Asunto(s)
Anhidrasa Carbónica II , Protones , Humanos , Bicarbonatos , Dióxido de Carbono , AguaRESUMEN
Hydrogen atoms are at the limit of visibility in X-ray structures even at high resolution. Neutron macromolecular crystallography (NMX) is an unambiguous method to locate hydrogens and study the significance of hydrogen bonding interactions in biological systems. Since NMX requires very large crystals, very few neutron structures of proteins have been determined yet. In addition, the most common hydrogen isotope 1H gives rise to significant background due to its large incoherent scattering cross-section. Therefore, it is advantageous to substitute as many hydrogens as possible with the heavier isotope 2H (deuterium) to reduce the sample volume requirement. While the solvent exchangeable hydrogens can be substituted by dissolving the protein in heavy water, complete deuterium labelling - perdeuteration - requires the protein to be expressed in heavy water with a deuterated carbon source. In this work, we developed an optimized method for large scale production of deuterium-labelled bacterial outer membrane protein F (OmpF) for NMX. OmpF was produced using deuterated media with different carbon sources. Mass spectrometry verified the integrity and level of deuteration of purified OmpF. Perdeuterated OmpF crystals diffracted X-rays to a resolution of 1.9 Å. This work lays the foundation for structural studies of membrane protein by neutron diffraction in future.
Asunto(s)
Deuterio/química , Escherichia coli/genética , Difracción de Neutrones/métodos , Neutrones , Porinas/química , Difracción de Rayos X/métodos , Chlorophyta/química , Chlorophyta/crecimiento & desarrollo , Clonación Molecular , Mezclas Complejas/química , Cristalografía por Rayos X/métodos , Medios de Cultivo/química , Medios de Cultivo/farmacología , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Modelos Moleculares , Porinas/genética , Porinas/aislamiento & purificación , Porinas/metabolismo , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Abstract: The hydrogen bond (H bond) is one of the most important interactions that form the foundation of secondary and tertiary protein structure. Beyond holding protein structures together, H bonds are also intimately involved in solvent coordination, ligand binding, and enzyme catalysis. The H bond by definition involves the light atom, H, and it is very difficult to study directly, especially with X-ray crystallographic techniques, due to the poor scattering power of H atoms. Neutron protein crystallography provides a powerful, complementary tool that can give unambiguous information to structural biologists on solvent organization and coordination, the electrostatics of ligand binding, the protonation states of amino acid side chains and catalytic water species. The method is complementary to X-ray crystallography and the dynamic data obtainable with NMR spectroscopy. Also, as it gives explicit H atom positions, it can be very valuable to computational chemistry where exact knowledge of protonation and solvent orientation can make a large difference in modeling. This article gives general information about neutron crystallography and shows specific examples of how the method has contributed to structural biology, structure-based drug design; and the understanding of fundamental questions of reaction mechanisms.
Asunto(s)
Sustancias Macromoleculares/química , Modelos Moleculares , Neutrones , Cristalografía por Rayos X , Enzimas/química , Enzimas/metabolismo , Humanos , Hidrógeno/química , Enlace de Hidrógeno , Hidróxidos/química , Ligandos , Conformación Molecular , Compuestos Onio/química , Oxidación-Reducción , Unión Proteica , Proteínas/química , Proteínas/metabolismo , Solventes/química , Especificidad por Sustrato , Agua/químicaRESUMEN
Glycoside hydrolase (GH) enzymes apply acid/base chemistry to catalyze the decomposition of complex carbohydrates. These ubiquitous enzymes accept protons from solvent and donate them to substrates at close to neutral pH by modulating the pKa values of key side chains during catalysis. However, it is not known how the catalytic acid residue acquires a proton and transfers it efficiently to the substrate. To better understand GH chemistry, we used macromolecular neutron crystallography to directly determine protonation and ionization states of the active site residues of a family 11 GH at multiple pD (pD=pH+0.4) values. The general acid glutamate (Glu) cycles between two conformations, upward and downward, but is protonated only in the downward orientation. We performed continuum electrostatics calculations to estimate the pKa values of the catalytic Glu residues in both the apo- and substrate-bound states of the enzyme. The calculated pKa of the Glu increases substantially when the side chain moves down. The energy barrier required to rotate the catalytic Glu residue back to the upward conformation, where it can protonate the glycosidic oxygen of the substrate, is 4.3 kcal/mol according to free energy simulations. These findings shed light on the initial stage of the glycoside hydrolysis reaction in which molecular motion enables the general acid catalyst to obtain a proton from the bulk solvent and deliver it to the glycosidic oxygen.
Asunto(s)
Proteínas Fúngicas/química , Glicósido Hidrolasas/química , Glicósidos/química , Neutrones , Biocatálisis , Conformación de Carbohidratos , Dominio Catalítico , Cristalografía por Rayos X , Proteínas Fúngicas/metabolismo , Ácido Glutámico/química , Ácido Glutámico/metabolismo , Glicósido Hidrolasas/metabolismo , Glicósidos/metabolismo , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Hidrólisis , Modelos Químicos , Modelos Moleculares , Unión Proteica , Estructura Terciaria de Proteína , Protones , Electricidad Estática , Especificidad por Sustrato , Temperatura , Trichoderma/enzimologíaRESUMEN
Human carbonic anhydrase II (HCA II) uses a Zn-bound OH(-)/H2O mechanism to catalyze the reversible hydration of CO2. This catalysis also involves a separate proton transfer step, mediated by an ordered solvent network coordinated by hydrophilic residues. One of these residues, Tyr7, was previously shown to be deprotonated in the neutron crystal structure at pH 10. This observation indicated that Tyr7 has a perturbed pKa compared with free tyrosine. To further probe the pKa of this residue, NMR spectroscopic measurements of [(13)C]Tyr-labeled holo HCA II (with active-site Zn present) were preformed to titrate all Tyr residues between pH 5.4-11.0. In addition, neutron studies of apo HCA II (with Zn removed from the active site) at pH 7.5 and holo HCA II at pH 6 were conducted. This detailed interrogation of tyrosines in HCA II by NMR and neutron crystallography revealed a significantly lowered pKa of Tyr7 and how pH and Tyr proximity to Zn affect hydrogen-bonding interactions.