Molecular Physiology: Enzymes and Cell SignalingProteinsBindingProtein SpecificityEnzymesEnzymatic ReactionsEnzyme ActivityEnzyme ConcentrationSubstrate ConcentrationSlide 10Catalytic EffectivenessAffinity for the SubstrateBinding of Additional LigandsRegulation of Enzyme ActivityPossible Ways of Controlling Gene ExpressionGene Regulatory ProteinsSlide 17Ligand Function in Gene RegulationModifying Activity of Existing EnzymesFeedback InhibitionAllosteric ModificationSlide 22Protein Phosphorylation (Covalent Modification)Cell SignalingMethods of Signaling Between CellsReceptor FunctionSignaling Cascade FunctionSlide 28Signaling PathwaysHydrophobic SignalsHydrophilic SignalsIon Channel-Linked ReceptorsG-protein-linked receptorsEnzyme-linked receptorsMolecular Physiology:Enzymes and Cell SignalingProteins•Polymers of amino acids•Have complex 3D structures•Are the basis of most of the structure and physiological function of cellsBinding•Much of protein function involves binding ligands–Ions, small molecules, macromolecules, etc.•Form noncovalent bonds with ligands–Individual bonds weak–Stable binding requires multiple bonds to be formedProtein Specificity•Proteins and ligands come together through random collisions•incorrect ligand –few bonds form, ligand dissociates quickly•correct ligand –multiple bonds form, ligand and protein remain associated for a long time intervalEnzymes•Protein catalysts•Bind ligand and chemically alter the ligand–Transform substrates into products•Lower activation energy of reactions–Increase reaction rate, allow non-spontaneous reactions to occur•Are not permanently altered in the reaction•very specific in the reactions they catalyze – hexokinase adds PO4 to D-glucose but not L-glucoseEnzymatic ReactionsEnzyme + Substrate  Enzyme-Substrate Complex  Enzyme-Product Complex  Enzyme + ProductEnzyme Activity•Ability of enzyme to convert substrate into product (i.e. reaction rate)•Key factors influencing enzyme activity1. Enzyme Concentration2. Substrate Concentration3. Catalytic Effectiveness4. Affinity of the enzyme for the substrate5. Binding of additional ligandsEnzyme Concentration•Increased concentration = more points for catalysis to take place•Proportional (linear) increase in reaction rate with increasing enzyme concentration[E]Reaction RateSubstrate Concentration•Hyperbolic or sigmoidal relationship•low [S], low reaction rate  [S],  rate •As [S],  % of enzyme molecules occupied in E-S complexes at a given time•Eventually, enzyme is saturated with substrate–No further  rate w/  [S]Substrate Concentration•Vmax–maximum velocity of the reaction–velocity at the point when enzyme is saturated with substrate•Vmax depends on…–Concentration of enzyme–Catalytic effectiveness of the enzymeCatalytic Effectiveness•The ability an enzyme molecule to rapidly convert bound substrate into product•Expressed by an enzyme’s turnover number (kcat)–# substrate molecules an enzyme molecule can convert into product per second•Influenced by1. The extent activation energy is lowered by the enzyme2. Time course of required changes in enzyme conformation (shape)Affinity for the Substrate•Ability of enzyme to effectively bind with colliding substrate•Michaelis-Menton constant (KM)–[S] at which ½ Vmax is achieved –a measure of an enzyme’s affinity for a substrate –( KM,  affinity)Binding of Additional Ligands•Enzyme may have additional binding sites for other ligands•Binding of ligands may influence enzyme shape–Influences substrate affinity and catalytic effectivenessRegulation of Enzyme Activity•Enzymes are KEY to controlling physiological function–Regulate ability of enzyme to catalyze reactions, regulate biochemical function•Enzyme activity can be controlled by1. Regulating the amount of enzyme present (regulating gene expression and enzyme degradation)2. Regulating the affinity and catalytic effectiveness of existing enzyme moleculesPossible Ways of Controlling Gene Expression1. control of transcription –how/when a gene is transcribed into RNA2. control of primary transcript processing and transport to the cytosol–splicing, etc.3. control of translation–selecting which mRNAs are translatedGene Regulatory Proteins•Repressors –proteins that bind DNA and block transcription •Activators –proteins bind to promoters that are only marginally functional in binding RNA polymerase alone–Enable RNA polymerase to bind•Function influenced by concentrations of specific ligands•eukaryotic transcription involves…–Transcription factors–Activators and repressors–Moderator complexes–Nucleosome proteins•Any/all can be affected by the specific concentration of various ligandsGene Regulatory ProteinsLigand Function in Gene Regulation•Example: Glucocorticoid Receptor–bonds glucocorticoid•Steroid hormone produced by adrenal glands–activates multiple genes for increased glucose production in the liverModifying Activity of Existing Enzymes•Metabolic pathways–Long chains of linked enzyme-catalyzed reactions•Require coordinated function of the enzymes–Reaction rates (enzyme activity) must be regulated–Regulated by controlling enzyme-substrate affinity and/or catalytic effectivenessFeedback Inhibition•Feedback Inhibition–enzyme activity inhibited by binding of a product from a subsequent reaction–As [Pfinal] ‘s, enzyme affinity ‘s, and Pfinal production ‘sAllosteric Modification•Allosteric enzymes–can assume > 1 shape •Enzyme may have multiple binding sites (active site + site for regulating ligand)•Binding of ligand alters shape of enzyme, altering affinityRegulation of Enzyme Activity•Positive Regulation–enzyme activity increased by binding of a product from a subsequent reaction As [Pfinal] ‘s, enzyme affinity ‘sProtein Phosphorylation(Covalent Modification)•Common method of altering shape of allosteric enzymes and other proteins–PO42- has large influence on protein shape–Reversible•protein phosphorylation catalyzed by protein kinases•dephosphorylated by protein phosphatasesCell Signaling•Animal cells need to coordinate their activities in order to maintain homeostasis•Must communicate chemically with one another to influence each other’s physiologyMethods of Signaling Between Cells1. Endocrine signaling–Broadcast to entire organism2. Paracrine