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This 5 page Class Notes was uploaded by Udbluehen03 on Saturday September 17, 2016. The Class Notes belongs to BISC401 at University of Delaware taught by Lachke,Salil in Fall 2016. Since its upload, it has received 10 views. For similar materials see Molecular Biology of the Cell in Biology at University of Delaware.
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Date Created: 09/17/16
Class 5 – 9/12/2016, Nucleic Acids Dr. Salil Lachke th Molecular Cell Biology, 8 edition – Lodish Proteins function depends on specific interactions with other molecules - Two proteins bind through interactions between their complementary surfaces Protein Binding and Enzyme Catalysis - The molecule in which a protein binds is called its ligand o The binding of a ligand to a protein is mediated by weak bonds o The binding site on a protein for a ligand consists of a number of amino acid residues brought together in the folded protein o A number of weak but specific interactions occur between the protein and ligand - Causes a change in the shape of a protein The properties of a protein that characterize it binds to a Ligand 1. Specificity o Ability of a protein to bind to molecules or a very small group of molecules in preference to all other molecules 2. Affinity o Refers to the tightness/strength of binding o Usually expressed as the dissociation constant (K ) d o K – dnverse of the equilibrium constant K . Theeqost common quantitative measure of affinity o The stronger the interaction between a protein and a ligand, the lower the valve of Kd Enzymes - Proteins that catalyze chemical reactions - The ligands of enzyme are called substrates - Most are located within the cells. Some are secreted and function at the extracellular sites such as blood. - Like all catalysts, enzymes increase the rate of a reaction, but they do not affect the extent of a reaction Class 5 – 9/12/2016, Nucleic Acids Dr. Salil Lachke Molecular Cell Biology, 8 edition – Lodish An Enzyme’s Active Site Binds Substrates and Carries out Catalysis - Active Site o Makes up only a small part of the total protein o Consists of the functionally important regions o 1. Substrate-binding site Recognizes and binds the substrate(s) Responsible for the remarkable specificity of enzymes Alteration of the structure of an enzyme’s substrate can result in a variant molecule that is no longer a substrate of the enzyme Only one or a few substrates can fit precisely into a binding site The binding site for a substrate on an enzyme can be exposed by the action of a small molecule o 2. Catalytic site Carries out the chemical reaction once the substrate has bound - The catalytic groups in the catalytic site are amino acid side chains and backbone carboxyl and amino groups - In some enzymes, the catalytic and substrate-binding sites overlap; in others, the two region are structurally distinct Serine Proteases - Large family of protein-cleaving - Used throughout the biological world; used to digest meal and control blood clothing - Trypsin, chymotrypsin, elastase How a Substrate Polypeptide Binds to the Substrate-binding site in the active site of Trypsin? - Two key binding interactions - 1. The substrate and enzyme form hydrogen bonds that resemble those of a β-sheet - 2. A Key side chain of the substrate that determines which peptide in the substrate is to be cleaved extends into the enzyme’s side-chain-specificity binding pocket, at the bottom of which resides the negatively charged side chai of trypsin’s Asp-189 Allostery - Refers to any change in a protein’s tertiary or quaternary structure, or in both, induced by the noncovalent bonding of a ligand Class 5 – 9/12/2016, Nucleic Acids Dr. Salil Lachke th Molecular Cell Biology, 8 edition – Lodish - When a ligand binds to one site (A) in a protein and induces a conformational change that alters the activity of a different side (B) o Ligand – allosteric effector/factor o Site A or site of ligand binding = allosteric binding site o The protein is called allosteric protein (target) - Important in feedback inhibition - Allosteric proteins have multiple binding sites, at least one for the allosteric effector and at least one for other molecules with which the protein interacts o The allosteric change in activity can be positive and negative o It can be an increase or decrease in protein activity Cooperativity - Often used synonymously with allostery - Refers to the influence that the binding of a ligand at one site has on the binding of another molecule of the same type of ligand at a different site - For example, hemoglobin o An example of positive cooperative binding o Binding of oxygen increases the affinity of hemoglobin for the next oxygen molecule o Four subunits in hemoglobin contains one heme molecule Heme groups are the oxygen binding components of hemoglobin - Amplifies the sensitivity of a system to changes in the concentration of its ligands, providing in many cases a sensitive evolutionary advantage Noncovalent Binding of Calcium - Widely used as allosteric - The concentration of free Ca in the cytosol is kept very low (≈10 M) -7 - Many Ca - binding proteins bind Ca using the EF hand/helix-looping-helix structural motif Calmodulin - Found in all eukaryotic cells - May be individual monomeric protein or a subunit of a multimeric protein 2+ - Very sensitive to Ca concentrations and changes in structure and function in response to Ca levels>10 M -7 Class 5 – 9/12/2016, Nucleic Acids Dr. Salil Lachke th Molecular Cell Biology, 8 edition – Lodish - Contains four Ca - binding EF hands with K of abdst 10 M -6 - The binding of Ca to calmodulin causes a conformational change that permits 2+ Ca /calmodulin to bind to conserved sequences in various target proteins. Noncovalent Binding of GTP - GTPase superfamily - Used as allosteric switches to control protein activity - They are enzymes – GTPases – that can hydrolyze GTP (guanosine triphosphate) to GTP (guanosine diphosphate) o The GTP is obtained from the cytosol - Include the monomeric Ras protein - Both Ras and Gα can bind to the plasma membrane - Functions o Cell signaling o Cell proliferation and differentiation o Protein synthesis o Transport of proteins between the nucleus and the cytoplasm o Vesicles - Some GTPase proteins have a covalently attached lipid chain that meditates their binging to membranes - Exist in two forms o 1. Active (on) form with bound GTP Influence the activity of specific target proteins to which they bind Intrinsic activity of GTPase is low o 2. Inactive (off) form with bound GDP - The switch is turned on o when a GTP molecule replaces a bound GDP in the inactive form - The switch is turned off o when the relatively slow GTPase activity of the protein hydrolyzes bound GTP, converting it to GDP and leading the conformation to change to the inactive form - The amount of the time GTPase switch remains in the active, GTP-bound form depends on the rate of its GTPase activity - GTPase-activating proteins (GAPs) increases the rate of GTPase activity, thus reducing the time the GTPase is in the active form Class 5 – 9/12/2016, Nucleic Acids Dr. Salil Lachke Molecular Cell Biology, 8 edition – Lodish o Activates GTPase activity (Ras, G alpha protein). Activation of GTPase activity results in GTP conversion to GDP. GDP-associated Ras is now inactive - Guanine nucleotide exchange factors (GEFs) o Proteins whose function is to regulate the conversion of inactive GTPases to active ones o Mediates exchange of GDP for GTP Phosphorylation and dephosphorylation - Covalently regulate protein activity - Phosphorylation o The reversible addition of phosphate groups into hydroxyl groups on the side chains of serine, threonine and tyrosine residues - Phosphoproteins o Phosphorylated proteins - Phosphorylation is catalyzed by enzymes called kinases - Dephosphorylation is catalyzed by phosphatases o Removal of phosphates - Phosphorylation changes a protein’s charge o Sometimes its shape o Which can result in conformational changes - Phosphorylation/dephosphorylation o Can influence the location of a protein within the cells o Its intrinsic activity o Its ability to bind to other molecules o Its ability to undergo further covalently modification o Its stability - Phosphorylation sites can be masked by NAG (N-acetyl glucosamine) - Nearly 3% of all yeast proteins are protein kinases/phosphates - In human genome there are about 500 human proteins kinases
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