CHEM 451 1st Edition Lecture 4Conformational Degrees of Freedom- Ex.) Ribosome: Large subunit has two “arms” that hinge for mechanical motion – GAC (GTP-activating center) can rotate up to 4 angstroms- Enzymes are a “mechanical scafold” – built to have certain hinges; conformational dynamics allow for specific large and small scale movement involved in catalysis; swiveling motions in the “head/neck”- Each reaction cycle of the ribosome incorporations one amino acid into the growing polypeptide chain; each incorporation = another swivel motionLigand-induced modulation of a diverse folding free energy landscapeOnce the enzyme folds into native state, it still has thermal motion embedded due to embedded energy (kBT Bolzmann constant)- Equilibrium exists between two diferent energy minima (two conformational states, e.g. ribosome hinging) - ΔG > kBT: Very few molecules have sufficient energy to overcome energy barrier (Maxwell-Bolzmanndistribution)- Conformational change (allosteric interactions) may shift from less active to more active state o Which of the two is favored?- Botom of folding free energy landscape: Excursions exist between macroscopic states. - F o ldi n g fun ne ls h a ve a fr a c t a l di m e n sion – self - s i m il a r i t y at dif e rent le n gth s ca l e s- En e r g y s cales are co nn ected to t i me s cales: less e ne r g y = f a ster o c c u rre n c e- Rememb e r, th e se are mul t i - dime n s io na l l a nd s ca p es – t he re is more t ha n on e p a t h way to a n o t h er state w i t h more th a n on e tra n si t i o n s tate! (a cry s tal s t ruct u re i s a n averaged sn ap sh o t)Macroscopic States: ΔG > kBT- Allosteric changes – ligand induces conformational change, shifting between macroscopic states (~μs) - Diference between less active and more active states- Consequence: more activity = more molecules spending time in active conformation - Examples: hinges on ribosomesMesoscopic States: ΔG ~ kBT- Molecules more readily traverse between diferent sub-states (~ns)- Examples: domain flopping (loop opening and closing); long-chain wobbling; twist of an α-helixMicroscopic States: ΔG < kBT- Even smaller energy barrier; rapid equilibrium (~ps)- Examples: movements of a single amino acid side-chain, hydrogen bond opening and closing, These notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.individual atomic motionsATCase as an example of dynamic allosteric controlATCase = aspartyl transcarbamoylase; in early stage of cytidine triphosphate synthesis; building block for RNA/DNA- First reaction (where flux is most): carbamoyl phosphate fuses with aspartate, catalyzed by ATCase to form carbamoyl phosphate- Enzyme is at the beginning of the pathway; hig h ly re gu l a ted by an activator and inhibitor (CTP)o End p r o d u ct i n h i b ition: transcription slows down, CTP accumulates, signaling for the first reaction to slow down (inhibits enzyme ATCase)o With CTP present, you need more aspartate to achieve the same relative reaction rateo ATP (energy currency is high, should be utilized) lowers the amount of aspartate needed, activating the cascade accelerating the reaction; cell may “decide to divide” if ample energy available-- This applies to ALL metabolic pathways – helps cell maintain activityHow does this work? Dynamic long-range interactions- T state (less active) and R state (reactive state) interconvert, depending on conditions o T state: open binding domainso R state: binding domains are closer togethero When hinge closes, the two substrates get closer together, making it easier to fuse - CTP favors T state, ATP favors R state- Allostery (combining of remote ligand) to activate enzyme changes the proportion of R and T state conformations------Ini t i a lly a cat al yt i c mo no mer, b u te n z yme f or ms a di mer. o T state has stericclashing of two subunitso R state has conformational rearrangement that allows the two subunits to slide togethero Advantage of dimers: cooperativity; conformational change of one subunit induces shift of otherThere are two branches of enzymology: kinetics and molecular mechanisms. K i n e t i c s : studying the speed of reactionsM ol ec u l a r Mec han i s m s : how molecular motions and electron movements within substrates catalyze reactionsA quick reminder: Chemical Kinetics- Measure the composition of the reaction mixture and how it changes over time. - Determine a rate law.- Move to a new solution with different concentrations of reactants and predict how quickly the reaction approaches the final state.- How do we do this?o Determine the stoichiometry and identify side reactionso Determine how the reagent and/or product concentrations change over time(measure initial concentration and continuously follow)-- Quench the reaction at defined times, keeping time constant -- Real-time analysis by spectroscopy- Flow method: continuous flow, change position of spectrometer to keep time constant - Stopped-fow method: mix two reagents in thechamber, spectrometer analyzessolution at one particular point, stop the flow and see how concentration of reagent