Allosteric signalingAllosteric mechanismsLigand induced conformationConcerted modelSequential modelGlucose metabolismPFKPEP inhibits PFKAllosteric homeostasis loop (PFK)Glucose storageGlycogen synthaseG6P regulation of GSCytoskeletal remodelingSmall GTPasesRho kinase, cytoskeletal remodelingGTPase cycleAllosteric signaling•Biochemistry•Direct negative feedback•Indirect feedback•Cyclic processesAllosteric mechanisms•Regulation by binding to a site other (άλλος allos) than the catalytic site•Multiple chemical states in complex molecules–Concerted, 2 state model–Sequential, multi-state model•Product – mediated feedback control•Oxygen-hemoglobin bindingLigand induced conformationDeoxy-hemoglobinOxy-hemoglobinGlobinHemeHeme-O2O2 allows pocket closureSmall shift in helixOne added AA interactionConcerted model•Equilibrium between distinct high and low affinity states•Multi-subunit molecules make a concerted or unified conformational change •Ligand binding increases the high affinity enzyme  cooperative binding•Monod et al., 1965Low affinitystateHigh affinitystate-2 -1 0 1 200.20.40.60.81Log (ligand)Bound Ligand Apparent affinity of H/L mixLigandSequential model•Equilibrium among multiple affinity states•Multi-subunit molecules undergo sequential conformational changes as each subunit binds ligand•Allows “Negative” cooperativity•Koshlind et al., 1966Low affinitystateHigh affinitystate-2 -1 0 1 200.20.40.60.81Log (ligand)Bound Ligand Apparent affinity of H/L mixLigandGlucose metabolism•Sequential phosphorylation of glocose•Symmetric cleavage to PEP & on to citric acid cycle•PFK is rate limitingPhospho-fructo-kinasePFKPFK•Activated by ADP, F1P•Inhibited by PEP (prokaryots) or citrate (eukaryots)PDB:4PFKPDB:3PFKAllosteric ADP binding siteActive siteNo reactants With reactantsPEP inhibits PFKPEP boundADP boundPEP binding causes a large scale reorganization of four monomers (Concerted model)Allosteric cofactor interacts with its own peptide chain and other subunit (green chain a; aqua chain b)PDB:6PFKPDB:4PFKAllosteric homeostasis loop (PFK)•Homeostasis: stable feedback controlControllerSensorPlantPFKPEPGlycolysisPFKADPGlycolysisGeneral modelPFK increases F1,6P, glycolysis converts this to PEP, which inhibits PFKPFK increases F1,6P, glycolysis converts ADP to ATP, reducing ADP, which is an activator of PFKGlucose storage•Extracellular glucose uptake•Phosphorylation by hexose kinase•Conversion to fructose 1,6,-bisphosphate•Storage in glyogen polymers–Conversion to UDP-glucose–Ligation by glycogen synthaseGlucosePhospho-glucosePhospho-fructoseFructosebisphosphateUDP-glucoseGlycogenGlycolysisHKisomeraseUDP glucosephosphorylaseGSPFKGlycogen synthase•Adds UDP-glucose to glycogen•Glucose-dependent•ATP-dependentGlucose-6-pGlycogenSynthaseGlycogenRothman-Denes & Cabib, 1971ControllerSensorPlantG6P is both substrate (via UDP-G) and regulator. Nonlinear dependence of rate on G6PG6P regulation of GS•Allosteric conformational changeWithout G6P With G6PBaskaran et al. 2010Cytoskeletal remodeling•Polymerizaton of actin filaments•Regulation of myosin contractility–Myosin Light Chain Kinase–Myosin Light Chain PhosphataseFocaladhesionRhoA ROCK MLP Cell motilitySmall GTPases•GTP is not usually a Pi donor•GTPases can be allosterically regulated allosteric regulators–GTPase timer–GAP switch•Guanine Activating Proteins (GAPs)–Facilitators of GTPase–Active of themselves–ie: GAPs may be allosterically regulated by GTP-GTPaseEF-Tu, the eEF1 homologRho kinase, cytoskeletal remodeling•GTP holds RhoA domains close•Residues of now adjacent domains bind ROCK1PDB:1S1CPDB:1FTNGDP-RhoA GTP-RhoA+ROCK1ROCK1GTPase cycle•GTP hydrolysis limits time of activation•Many GTPase effectors are GAPs–eg: ribosome–Autoinhibitory, self-sensing controller•Many GTPases require GEFs–Less a sensor of [GTP]–More a communication methodGEFGAPGDP-GTPaseInactiveGTP-GTPaseActiveGTP ExchangeGTP