BSC 450: Week 6 Notes
BSC 450: Week 6 Notes BSC 450
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This 12 page Class Notes was uploaded by Jordana Baraad on Friday October 2, 2015. The Class Notes belongs to BSC 450 at University of Alabama - Tuscaloosa taught by Dr. Ramonell in Summer 2015. Since its upload, it has received 71 views. For similar materials see Fundamentals of Biochemistry in Biological Sciences at University of Alabama - Tuscaloosa.
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Date Created: 10/02/15
Enzymes act as catalysts by stabilizing the transition state T5 of a reaction a No enzymes a Uncatalyzed b Highest activation barrier b Enzyme complementary to substrate a Lock and Key Model Emil Fischer 1890 b Intermediate activation barrier c Enzyme complementary to TS Induced fit Lowest activation barrier Ex Hexokinase some parts of the molecule that don t appear important actually are interact w enzyme in transition state contribute to bonding energy Enzymes use common catalytic mechanisms Acidbase chemistry give and take protons General v specific Specific hydronium and hydroxide ions change pH General primary mechanism we study in this class His classic Cys COOH Covalent catalysis change reaction paths Transient covalent bond w some intermediate Transfer intermediate to active site Hold it there OR attach to enzyme to make it a good leaving group Metal ion catalysis use redox cofactors pka shifters Lower pka of water Ex Zn water pka 15 9 quot7 Ex Heme group with Fe Fe and Cu most common Amino acids don t accept donate electrons Entropic reduction preferential interaction with TS Holding sites adjacent to one another Stuck in rigid position to force reaction Michaelis Menten equation l39 V K x not given on test PUT ON CARD derivation not necessary see Fig 611 2 part curve Part of curve that flattens max capacity Adding substrate doesn t increase reaction rate Early linear portion dependent on substrate concentaration Km 9 substrate concentration 12 Vmax Michaelis Menten assumptions 2 k1 k3 ES ES EP k2 k4 1 Only looking at initial times in product a Worried about ES 9 EP not reversible i Reversible binding followed by irreversible chemical step 2 ES ES happens quickly a Problem true for enzyme they were studying but not all b Laster revised 19205 9 steady state assumption Briggs amp Haldane Steady State Assumption Revision 9 modern version of MichaelisMenten equation If know vmax km and substrate concentration can calculate velocity at any point in rxn What Does km mean km yz Vmax NOT a velocity NOT a kD binding affinity We can now evaluate Michaelis Menten at low and high S At low S beginning quotlinearquot part of slope Reaction rate dependent on amount of substrate Slope vmax km Later part approaching vmax Flat line Can t change reaction rate by adding more substrate What Does vm m Tells about enzyme efficiency Perfect enzyme works quickly rapid binding In vmax km higher v rxn faster 9 better enzyme lower km need less substrate to get to same velocity compared to other enzymes 9 better enzyme Graphical Analysis Tabular Analysis 1 E mM mMs l E Z 2 E E E E E Q Q btwn S 20 and S 40 v starting to level off 9 vmas approx 90 mMs km at 12 vmax so at 45 mMs 5 mM Taking Reciprocal of MichaelisMenton equation line V0 V E VI39ldx y m X l b LineweaverBurk Plot aka Double Reciprocal plot Xintercept 1km Important for next set of materials How enzymes work Competitive inhibition Distinct patterns for different mechanisms What Do We Need to Know to Define an Enzymatic Mechanism Sequence More than 1 product Multiple enzyme forms Most enzymes have multiple substrates and products Structure of intermediates TS Rate of conversion Structural relations What amino acids in active site are involved in rxn Postion Interation with water Energy contribution Contribution of each reacting group overall Enzymes core of biochem research Particularly drug design Know whole pathway then go back to target intermediates Cleland Notation Wallace Cleland quotgrandfather of enzymology ES ES EP EP Substrates A B C Products P Q R Inhibitors I J K Enzyme Forms E F Ex ls 1p uni uni reaction What about multi substrate enzymes Simple doesn t tell us how products bind or leave 1 Ordered strict order of binding 2 Random flexible enzyme both enzymes examples of 25 2p bi bi mechanisms both considered sequential regardless of order all substrates bound before chemistry occurs sequential NOT ordered Kinetics Can Identify x intercept 1km y int 1vmax m km Vmax early linear phase dependent on 5 later flat portion approaching or at vmaxz not depedent on 5 experiment hold 1 substrate constant add increasing amounts of the other Michael Mensen plot vmax tells how enzyme behaves at highincreasing 5 Linear slope tells how enzyme behaves at lowdecreasing S Lineweaver Burke plot y intercept tells how enzyme behaves when 5 increasing Slope tells how enzyme behaves when S decreasing Sequential Kinetic Mechanism see figure 6 1a intersection pattern lines intersect at single point away from y axis indicator that enzymes acting in sequential mechanism Ping Pong Kinetic Mechanism Enzyme Inhibition 2 big classes enzyme inhibitors 1 irreversible inactivate poison enzyme aka suicide inhibitor a tag to recycle amino acids can t ever use actual enzyme again b covalent linkage highly reactive c ex powerful toxins venom drugs 2 reversible inhibitors bind and dissociate a competitive b uncompetitive c noncompetitive ex drugs that tone down as opposed to shutting off enzyme activity structural analogs of substrates products Competitive Inhibition Looks like substrate binds at active site LineweaverBurke plot all lines intersect yaxis quotnumbers game if 5 gtgt I little change if more I 9 increased chance of inhibition Uncompetitive Inhibition ONLY binds to ES complex No place for I until ES forms Once I binds no product formation Slopes stay same like pingpong mechanism Difference varying I Noncompetitive Inhibition better name Mixed Inhibition know both powerful binds w free enzyme AN DOR ES complex Lineweaver Burke plot 3 y intercepts away from y axis Intersecting pattern For test know how to interpret inhibition Or plain mechanism Irreversible Transition state TS analogs 9 best class of irreversible inhibitors Look very similar to TS Take advantage of EVERY weak interaction possible Tight binding to enzyme Block any chemistry Very low kd dissociation constant Enzyme activation can be regulated noncovalent covalent transferring ex transfer remove phosphate group irreversible reversible quotonoff switch common in cells often combination regulation multiple levels redundancy bc regulated enzymes are the most important vital to life Controlling Protein Function Not just regulated at protein level Products 9 feedback inhibition Regulation types Expression Degradation particular domains indicate short life span Tagged with ubiquitin quickly Sent to proteasome for degradation Proteasome can be confined to particular domains More common in plants ie vacuole Typically held by modification eg add sugar Cleaving modification 9 functionality Change protein conformation Add phosphates allosteric inhibition Change shape 9 modification Can be positively or negatively regulated Allosteric Enzymes Can be positively or negatively regulated Pics rt open active site regular molecule CTP allosteric modification binds away from active site left 4 bound CTPs 9 shutdown can t bind substrates effective common quotonoff switch result Phosphorylation Another common quotonof regulation mechanism Add phosphate w kinase ON Much more studied Remove phosphate w phosphatase OFF Understudied Most commonly phosphorylated amino acids Ser Thr Tyr Induce shape change
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