Bio 285 SI Before Exam 2 March 6Mechanism of Enzyme Function:- Q: What do enzymes do to a reaction? (energy of the graph)o They lower the activation energy and the transition stateo Peak of graph is transition state – lowerso Speeds up rate of reaction- Enzymes are catalysts- Do we change free energy of reactants and products? NO- If energy of the reactants is higher than products = exergonico Released energy, so molecule is STABLE- If energy of products is higher than reactants = endergonico Considered UNSTABLE- Enzymes take a substrate, form a complex, then switch substrate into product, then release producto 3 ways to do this: Enzyme binds to 2 substrate molecules and orients them precisely to encourage a reactionto occur between them Binding of substrate to enzyme rearranges electrons in the substrate, creating partial negative and positive charges that favor a reaction Enzyme strains the bound substrate molecule, forcing it toward a transition state to favor a reactiono Enzyme with substrate has higher binding affinity b/c wants to let product go, needs to hold onto substrate to be able to turn it into product- Q: pull down assay effective way to look for substrates?o Reaction takes a long time, so by that time substrate would be product so not useful- Enzymes lower the activation energy of a chemical reactionAllostery/Regulation of Enzyme/Protein Function:- Allostery – when something other than the substrate binds to somewhere other than the active site; causes conformational changeo Activate enzyme or inhibit enzyme- When allosteric enzyme binds, changes shape / functiono Can be positive or negative- Ex) Hemoglobino When one molecule of O2 binds to a hem group, changes conformation of the other 3 hem groups which then have a higher affinity to O2- Post-translational modifications – you modifying the protein after protein has been createdo Phosphorylation, ubiquitination, methylation, acetylation- Post-translational modify RTK through phosphorylation- Nucleotide binding/hydrolysis can lead to different conformational and active states- Hydrolyze GTP to GDP use GAP (inactive)- Kick out GDP to put GTP in use GEF (active)General Principles of Cell Signaling & Signal Transduction:- Cell is always receiving signals (survive, grow & divide, differentiate)- If cell doesn’t receive a signal, dies (programmed cell death) (apoptosis)- Cannot grow human cells in labs b/c need to be able to tell it to “SURVIVE” all the time- Two general outcomes for cell signaling when dealing with proteins:o Changing level of proteins (slower)o Changing the existing function of an existing protein (faster b/c don’t need to go through transcription, translation, etc. (more time to synthesize more proteins))Activating Receptor Tyrosine Kinases (RTKs):- *draw pathway out over and over until known by heart*- RTKs involved in both growth pathways & survival pathways- How activate RTKs?o Two subunits of RTK separatedo Signal molecules that forms a dimer (dimerization = two subunits come together and form a functional unit)o Intrinsic kinase activity activated / kinase activity stimulated b/c dimerizedo Add phosphates through cross-phosphorylationo (no keys, can’t start car, car can’t move; no gas, car can start, car can’t move) (difference is car CAN start in second scenario) If can’t phosphorylate the sites, dimerization can happen because phosphorylation happens after dimerization If can’t dimerize, can’t phosphorylate because dimerize happens before phosphorylationo Adding phosphates to tyrosine on receptors – causes receptors to be activeo Then, other molecules/proteins bind to those phosphorylated tyrosines for pathway to happen These proteins have SH2 domain (SH2 domain binds only to phosphotyrosine residues – will NOT bind to any other amino acid)o Then, go onto the Ras pathwayMechanism of Ras Signaling:- Continues from above- Adaptor protein (Grb2) binds to a phosphorylated tyrosine- Ras-activating protein (SOS) binds to Grb2- Grb2 activates Ras-activating protein- Ras-activating protein looks for an inactive Ras protein- Kicks out GDP in inactive Ras, allows for GTP to bind in Ras (to become active) (acts as a GEF)- Ras now active goes to find a MapKKK kinase- Activates MapKKK --- MapKKK goes and activates MapKK by exchanging an ATP for an ADP – needs 2 phosphates, get one phosphate from ATP and already had a phosphate- MapKK goes and activates MapK by exchanging an ATP for and ADP – needs 2 phosphates, get one phosphate from ATP and already had a phosphateo MapKKK is different than MapKK is different than MapK – NOT the same kinase at allo Activating each other = Map kinase cascade- MapK goes and activates protein X and protein Y OR transcription regulated A and transcription regulator B by giving them phosphateso Proteins X and Y cause a change protein activityo Transcription factors A and B cause a change gene expressionSurvival Signal Pathway that go through RTK:- End game is to prevent cell death/apoptosis- Get a signal that causes dimerization that causes cross-phosphorylation for receptor- Tyrosines phosphorylated, PI3 kinase binds to phosphotyrosines – has an SH2 domain- PI3 kinase activates inositol phospholipid by adding another phosphate to it (goes from 2 phosphates to 3 phosphates = active)- Phosphorylated inositol phospholipid (now has 3 phosphates) creates a binding site for AKT and protein kinase 1- Protein kinase 1 attached to inositol phospholipid and AKT attached to another active (3 phosphates) inositol phospholipid o Need 2 binding sites – 1 for AKT and 1 for protein kinase 1- Protein kinase 1 and protein kinase 2 (another protein kinase) add a phosphate to AKT to make it active (need 5 phosphates to be active) (shares 3 phosphates with inositol phospholipid when binds to it, gets 2 more phosphates from protein kinase 1 (1) and protein kinase 2 (1))- Once add two phosphates to AKT, AKT disassociates from inositol phospholipid and goes to target Bado Bad inhibits Bcl2 ( = cell death) (causes Bcl2 to not do its function = cell going to die) (Bad activewhen bound to Bcl2) (Bcl2 inactive when bound to Bad)o Bcl2 inhibits cell death ( = no cell death)- AKT phosphorylates (adds 1) Bad, changes conformation of it, and that causes Bad to release Bcl2 (causes Bad to become inactive) (causes Bcl2 to become active)- Inactivated Bad goes off, Bcl2 now active so goes to cause cell death- Q: can’t add a phosphate to AKT = can’t