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Ch12-120323 - CHEM 350 Introduction to Biological...

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Unformatted text preview: CHEM 350: Introduction to Biological Chemistry! "[email protected]! "Brian Lee, Ph.D. ! "Office: Neckers 146G or 324 "Phone: 453-7186! "Hours: 9:30am to 10:30am or by appointment! "Website: https:/ /online.siu.edu! Textbook (required, U.S. edition only)! Fundamentals of Biochemistry, 3rd Ed., Voet, Voet & Pratt. ! Study Guide (recommended)! Student Companion to Fundamentals of Biochemistry, 3rd Ed. ! Help Desk! Tuesday 6:30 to 7:30 pm in Neckers 218! Thursday 5:00 to 6:00 pm in Neckers 410! Announcements! Undergraduate Research Opportunities! Research for credit (such as CHEM 396 or CHEM 496)! Student worker ($8.00 per hour) (http://www.siu.edu/~fao/jobs/)! Undergraduate Assistantships (http://undergraduateassistantship.siuc.edu/)! McNair Scholars Program (http://www.siu.edu/~mcnair)! REACH Awards Competition (http://www.siu.edu/~reach/) (due Jan 30t ! ! Assignments! •! Read 12 Enzymatic Kinetics and Chapter 12 Problems! •! Third MidTerm Exam - Wednesday, March 28th! •! Chapters 10-13 (Chapter 13: Sections 1, 2A and 2B)! ! ! Help Desk! Tuesday 6:30 to 7:30 pm in Neckers 218! Thursday 5:00 to 6:00 pm in Neckers 410! Michaelis-Menten Equation! k1 k2 E + S " " # ES " " # P + E " " k-1 ! Vmax [S] v0 = K M + [S] KM is Michaelis constant! k"1 + k2 KM = k1 KM is the substrate concentration at which the reaction velocity is half maximal.! v0 1 [S] == Vmax 2 K M + [S] KM = k"1 k 2 k + = KS + 2 k1 k1 k1 KS is the dissociation constant of the Michaelis complex, ES -> E + S! ! As KS decreases, the enzymes affinity for substrate increases! Catalytic Efficiency! k1 k2 E + S " " # ES " " # P + E " " k-1 ! At high substrate concentration:! kcat Vmax = k2 = [E]T Vmax [S] v0 = K M + [S] v 0 = Vmax = k 2 [ E ]T kcat is the turnover number! At low substrate concentration:! v0 = k cat [E]T [S] KM Diffusion-controlled limit! kcat/KM = k1 = 108 to 109 M-1s-1! kcat/KM measures efficiency! Kinetics of Enzyme Inhibition! Competitive Inhibitor! -! increase [S] to recover! -! apparent KM increases! -! no change in Vmax! Mixed Inhibitor (affects [E])! - no effect with increased [S]! - apparent Vmax decreases! - no change in KM (if KI(E)=KI(ES))! Confusion between uncompetitive and non-competitive?! ! Uncompetitive inhibitors ambush the substrate.! They cheat and let the substrate bind to the enzyme first.! ! Pure non-competitive inhibitors don t care about substrate.! They do not compete, they just bind to the enzyme.! ! ! Mixed (non-competitive) inhibitors bind to the enzyme or the enzyme-substrate complex with different affinities. ! Mixed inhibition with ! = ! is also called:! pure non-competitive inhibition.! Product Inhibition! If release of fumarate! is slow, then fumarate! will prevent binding of ! succinate.! ! Product inhibition is one! way to regulate enzyme! activity in response to ! product concentration.! $" $# slow! " " " " !! + # " # "# " # "% " # % + " k-1! Product inhibition of adenosine deaminase:! KM = 3 x 10-5 M! Transition state analog! KI = 1.5 x 10-13 M! KI = 3 x 10-4 M! Transition state analogs are used to study! the mechanism of the enzyme reaction.! Phosphonate ester as a! mimic of the tetrahedral! carbon transition state.! ! Enzymes increase the rate of catalysis by stabilizing the substrate in the transition state.! Transition state analogs will bind tighter than ! the native substrate.! Irreversible Inhibitors are often used to identify! active site residues that participate in catalysis! Chymotrypsin identified as a! serine protease by DIPF! DIPF was original found! to be potent nerve! agent that inactivates! acetylcholinesterase ! (a serine esterase)! DIPF = diisopropylphosphofluoridate! Affinity labeling uses a substrate analog to tag an active site residue that can be later identified by peptide mapping or amino acid analysis! Alkylation of His57 in Chymotrypsin! Suicide inhibitor requires the enzyme to ! activate the inhibitor prior to modification! Enzyme oxidizes! inhibitor! Inhibitor then! alkylates flavin! coenzyme! Regulation of Enzyme Activity! regulatory enzymes - subject to control! ! allosteric regulation - regulation by small molecules! (modulators or effectors) that bind at a different site.! ! ! homotropic effects - i dentical ligand and effector! heterotropic effect - different ligand and effector! mechanisms of regulation:! covalent modification - switching on and off! regulatory complex - enzyme and regulator subunit! proteolytic activation - removal of inhibiting segments! synthesis and degradation - response to environment ! Catalytic subunit! Regulator subunit! Switch between T and R states! Allosteric Regulation! Allosteric enzymes do not! follow Michelis-Menten! kinetics. The saturation! curve is sigmoidal rather! than rectangular hyperbolic.! Aspartate transcarbamoylase (ATCase)! Pyrimidine! Biosynthesis! Feedback Inhibition of ATCase by CTP! CTP is an allosteric ihibitor of ATCase! ATP is an allosteric activator of ATCase! PALA is a substrate analog that binds to the R state! PALA does not react, but stabilizes the R state! Used to study the R state conformation.! Page 388 ATCase has 12 subunits! 6 catalytic (red/blue)! 6 regulator (yellow)! dimer of trimers! T state! R state! Figure 12-12 R T! Transition from T to R state brings substrates together! CTP! ATP! Regulation by! covalent modification! ! Phosphorylation! -kinase! -phosphatase! Page 390 Glycogen phosphorylase! glycogen( n ) + Pi " glycogen ( n # 1) + G1P Breakdown of glycogen! for energy metabolism.! ! ! Regulated by phosphorylation! of glycogen phosphorylase.! Ser 14 is phosphorylated.! Phosphorylase a dimer! -active site (pyridoxal phosphate)! -allosteric effector site! -glycogen binding site! -catalytic site! Phosphorylation stabilizes the R state through favorable! interactions with the Ser14 phosphate group by Arg569! Phosphorylation also changes which allosteric regulators! are recognized by phosphorylase:! phosphorylase b ( ATP, G6P, AMP) ! phosphorylase a (glucose)! ...
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