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Chapter 5&6

Chapter 5 & 6.docx

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Wilfrid Laurier University
Geoff Horsman

Chapter 5: Protein Function Ligand -A molecule bound reversibly by a protein Binding site - A ligand binds at a site on the protein - Is complementary to the ligand in size, shape, charge, and hydrophobic or hydrophilic character - Different ligands may have separate binding sites for several Induced fit - The structural adaptation that occurs between protein and ligand is called - enzyme changes shape to fit better with the ligand when it binds to - In a multisubunit protein, a conformational change in one subunit often affects the conformation of other subunits Reversible ligand binding: oxygen-binding proteins - Oxygen is poorly soluble in aqueous solutions - Diffusion of oxygen through tissues is also ineffective over distances greater than a few millimeters. - However, none of the amino acid side chains in proteins is suited for the reversible binding of oxygen molecules - Heme is buried deep within protein to prevent irreversible oxidation of ferrous ion (simultaneous reaction of one O2 with two heme molecules will result in oxidation) Protein Structure Affects How Ligands Bind - the specificity with which heme binds its various ligands altered when the heme is a component of myoglobin. - Carbon monoxide binds to free heme molecules more than 20,000 times better than does O2 - Kd or P50 for CO binding to free heme is more than 20,000 times lower than that for O2 - Recall: the smaller the dissociation constant, the more tightly bound the ligand is, or the higher the affinity between ligand and protein. - but it binds only about 200 times better when the heme is bound in myoglobin - difference explained by steric henderence - When O2 binds to free heme, the axis of the oxygen molecule is at an angle to the Fe--O--O bond - In contrast, when CO binds to free heme, the Fe, C, and O atoms lie in a straight line - In myoglobin, His64 (His E7), on the O2-binding side of the heme, is too far away to coordinate with the heme iron, but it does interact with a ligand bound to heme. This residue, called the distal His, does not affect the binding of O2 ***( In other words, Oxygen binds to heme with the O2 axis at an angle, a binding conformation readily accommodated by myoglobin. Carbon monoxide binds to free heme with the CO axis perpendicular to the plane of the porphyrin ring. When binding to the heme in myoglobin, CO is forced to adopt a slight angle because the perpendicular arrangement is sterically blocked by His E7, the distal His. This effect weakens the binding of CO to myoglobin) - binding of O2 to the heme in myoglobin also depends on molecular motions, or “breathing,” in the protein structure - One major route for O2 to have access to bind to Fe is provided by rotation of the side chain of the distal His (His64), which occurs on a nanosecond - erythrocytes are formed from precursor stem cells called hemocytoblasts - stem cell produces daughter cells that form large amounts of hemoglobin and then lose their intracellular organelles—nucleus, mitochondria, and endoplasmic reticulum. - Erythrocytes are thus incomplete, vestigial cells, unable to reproduce and, in humans, destined to survive for only about 120 days - release one-third of the oxygen in hemoglobin - Myoglobin, with its hyperbolic binding curve for Oxygen - relatively insensitive to small changes in the concentration of dissolved oxygen and so functions well as an oxygen-storage protein. - Hemoglobin, with its multiple subunits and O2-binding sites, is better suited to oxygen transport - more sensitive to small changes in concentration of oxygen - Hemoglobin is a tetrameric protein containing four heme prosthetic groups, one associated with each polypeptide chain - Adult hemoglobin contains two types of globin, two α chains (141 residues each) and two β chains (146 residues each) - fewer than half of the amino acid residues in the polypeptide sequences of the α and β subunits are identical, the three-dimensional structures of the two types of subunits are very similar - When oxygen binds, the α1β1 contact changes little, but there is a large change at the α1β2 contact, with several ion pairs broken - At α1β1 and α2β2 involves more than 30 residues, and its interaction is sufficiently strong - can disassemble into αβ dimers - At α1β2 and α2β1 , hydrophobic interactions predominate at the interfaces, but there are also many hydrogen bonds and a few ion pairs Binding equilibrium Hemoglobin Undergoes a Structural Change on Binding Oxygen - two major conformations of hemoglobin: the R state and the T state - Although oxygenbinds to hemoglobin in either state - higher affinity for hemoglobin in the R state - b/c Oxygen binding stabilizes the R state - T state is more stable when no oxygen and is thus the predominant conformation of deoxyhemoglobin - T state is stabilized by a greater number of ion pairs also called “tense” state - Binding of oxygen to hemoglobin leads the αβ subunit pairs slide past each other and rotate, narrowing the pocket between the β subunits -More planar heme resulting from O2 binding propogates structural changes to the F helix and leads to adjustments at the α1β2 interface - ion pairs that stabilize the T state are broken and some new ones are formed - allosteric protein is one in which the binding of a ligand to one site affects the binding properties of another site on the same protein - induced by the binding of ligands referred to as modulators - The modulators for allosteric proteins may be either inhibitors or activators - When the normal ligand and modulator are identical, the interaction is called homotropic - When the modulator is a molecule other than the normal ligand the interaction is heterotropic. - binding of O2 to hemoglobin, is a form of allosteric binding often observed in multimeric proteins. - considered homotropic, b/c ligand and activator is the same Two Models Suggest Mechanisms for Cooperative Binding - concerted model assumes that the subunits of a cooperatively binding protein are functionally identical, that each subunit can exist in two conformations, and that all subunits undergo the transition from one conformation to the other simultaneously - no protein has individual subunits in different conformations. The two conformations are in equilibrium. The ligand can bind to either conformation, but binds each with different affinity. Successive binding of ligand molecules to the low-affinity conformation (which is more stable in the absence of ligand) makes a transition to the high-affinity conformation more likely - sequential model - ligand binding can induce a change of conformation in an individual subunit - conformational change in one subunit makes a similar change in an adjacent subunit, as well as the binding of a second ligand molecule, more likely - main difference b/n models is that more potential intermediate states in this model than in the concerted model Hemoglobin Also Transports H+ and CO2 - hemoglobin carries two end products of cellular respiration—H+ and CO2— from the tissues to the lungs and the kidneys, where they are excreted - CO2, produced by oxidation oforganic fuels in mitochondria, is hydrated to form bicarbonate - reaction is catalyzed by carbonic anhydrase, an enzyme particularly abundant in erythrocytes - hydration of CO2 results in an increase in the H+ concentration (a decrease in pH) - binding of oxygen by hemoglobin is profoundly influenced by pH and CO2 concentration - effect of pH and CO2 concentration on the binding and release of oxygen by hemoglobin is called the Bohr effect - Oxygen and H+ are not bound at the same sites in hemoglobin - Oxygen binds to the iron atoms of the hemes, whereas H_ binds to any of several amino acid residues in the protein - When protonated His146 of the β subunits forms one of the ion pairs—to Asp94 that helps stabilize deoxyhemoglobin in the T state -Major contribution to the Bohr Effect made by His HC3 - The ion pair stabilizes the protonated form of His HC3, giving this residue an abnormally high pKa in the T state - pKais usually below 6 at R state and a oxyhemoglobin has a pH of 7.6, usually blood in lungs pH - Protonation of the amino-terminal residues of the α subunits, -four polypeptide chains of hemoglobin communicate with each other about not only O2 binding to their heme groups but also H+ binding to specific amino acid residues -The interaction of 2,3-bisphosphoglycerate (BPG) with hemoglobin provides an example of heterotropic allosteric modulation - BPG. 2,3-Bisphosphoglycerate is known to greatly reduce the affinity of hemoglobin for oxygen - At higher altitudes = partial pressure of O2 is lower = delivery of O2 is less = increase in the concentration of BPG = bind O2 the same at lung but decreases affinity= back to 40% transport of O2 - The site of BPG binding to hemoglobin is the cavity between the β subunits in the T state - lined with positively charged amino acid residues that interact with the negatively charged groups of BPG. -only one molecule of BPG is bound to each hemoglobin tetramer - BPG binds to hemoglobin and lowers hemoglobin’s affinity and stabilizes the T state; the R state with higher O2 affinity narrows and loses BPG binding pocket. - BPG plays important role in fetal development; fetal hemoglobin must have greater affinity for O2 than mother’s hemoglobin in order to extract O2 from mother’s blood. - Fetus makes (αγ)2 hemoglobin, which has lower affinity for BPG - hereditary human disease sickle-cell anemia from both parents - Most variations consist of differences in a single amino acid residue - the blood contains many long, thin, crescent-shaped erythrocytes that look like the blade of a sickle -hemoglobin from sickl
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