4.高级版课件2英文part i structural biochemistry-chapter9enzyme kinetics

1、Chapter 9 Enzyme KineticsOutlineDefinition of enzyme kineticsFactors affecting enzyme-catalyzed reactionsMichaelis-Menten KineticsEnzyme inhibition kinetics Allosteric enzyme kineticsIntroduction to Enzyme KineticsKinetics concerns with the rates of chemical reaction. Enzyme kinetics addresses the b

2、iological roles of enzyme and quantify the remarkable function of enzymes;Enzyme kinetics information can be exploited to control and manipulate the course of metabolic events. Pharmaceuticals or drugs are often special inhibitors targeted at a particular enzyme. Leonor Michaelis (1875-1949)Maud Men

3、ten (1879-1960)Enzyme KineticsThe rate of unimolecular reaction is proportional to the concentration of the reactant. Thus rate is linearily dependent on A. But if this reaction is catalyzed by an enzyme, the rate shows saturation behavior. Why?vAvAThe Mechaelis-Menten EquationYou need to know how t

4、his is derivedThis is the complete chemical formula for an enzyme-catalyzed (E) reaction of substrate, S and product, P;Mechaelis-Menten equation describes the relationship between reaction rate and substrate concentration. It can explain the saturation behavior in catalyzed reactions as shown in th

5、e previous slide. Mechaelis-Menten equation is derived based on the following three conditions:State steady assumption;Initial velocity assumption;Rate law.k1K-1k2K-2Initial Velocity AssumptionIn the beginning of the reaction, there is very little product, or P is small. So the amount of ES contribu

6、ted by E+P is negligible.Thus, the MM equation concerns the reaction rate that is measured during early reaction period. In which case, the enzyme catalyzed reaction can be modified to:K-1k1K-1k2k1k2K-2Rate Law in Enzyme Catalyzed ReactionsRate law still applies in enzyme catalyzed reactions. The fo

7、rward velocity, or rate, vf is,The reverse velocity or rate, or the rate of disappearance vd is,At steady state, there is no accumulation of ES, thus: k1K-1k2Derivation of Michaelis-Menten EquationWe need one more condition, that is, the total enzyme concentration, Et is the sum of that of enzyme-su

8、bstrate complex, ES, and that of free enzyme, E:At steady state, the forward rate should equal to the reverse rate: Rate of production formation (rate law), v = k2ES. So:Notes on the MM EquationsThe rate of production formation can usually be measured experimentally by monitoring the progress curve

9、of production formation. The maximum rate can be reached at saturating substrate concentration, or when SSo MM equation can be re-written as:Enzyme-catalyzed rate is saturatedUnderstanding KmKm is a constant derived from rate constants Km is, under true Michaelis-Menten conditions, an estimate of th

10、e dissociation constant of E from S, because at equilibrium, Reversible reaction, dissociation constant is So small Km means tight substrate binding; high Km means weak substrate binding.Km equals to the substrate concentration at which v=1/2vmaxk1k-1Understanding VmaxThe theoretical maximal velocit

11、y Vmax is a constant Vmax is the theoretical maximal rate of the reaction - but it is NEVER achieved in reality To reach Vmax would require that ALL enzyme molecules are tightly bound with substrate Vmax is asymptotically approached as substrate is increased The dual nature of the MM equationCombina

12、tion of 0-order and 1st-order kinetics When S is low, the equation for rate is 1st order in S When S is high, the equation for rate is 0-order in S The Michaelis-Menten equation describes a rectangular hyperbolic dependence of v on S!The turnover numberA measure of catalytic activity kcat, the turno

13、ver number, is the number of substrate molecules converted to product per enzyme molecule per unit of time, when E is saturated with substrate. If the M-M model fits, k2 = kcat = Vmax/Et Values of kcat range from less than 1/sec to many millions per secValues of kcat for Some EnzymesThe catalytic ef

14、ficiencyName for kcat/KmAn estimate of how perfect the enzyme is kcat/Km is an apparent second-order rate constant It measures how the enzyme performs when S is low The upper limit for kcat/Km is the diffusion limit - the rate at which E and S diffuse together The Values of kcat, Km and kcat/Km for

15、some enzymesA better method for determining the values of Vmax and Km, which was formulated by Hans Lineweaver and Dean Burk, uses the reciprocal of the MichaelisMenten equationDetermining the Vmax and Km Lineweaver-Burk or double-reciprocal plotEnzyme InhibitionEnzyme can be inhibited by inhibitors

16、. Inhibitors are tools to scientists to understand enzymes. Inhibitors are also in many cases pharmaceutical reagents against diseases;Inhibitors inhibit enzyme function by binding with enzymes. The binding reaction can be either reversible or irreversible;Reversible inhibitors associate with enzyme

17、s through non-covalent interactions. Reversible inhibitors include three kinds:Competitive inhibitors; petitive inhibitors; petitive inhibitorsIrreversible inhibitors associate with enzymes through covalent interactions. Thus the consequences of irreversible inhibitors is to decrease in the concentr

18、ation of active enzymes. Competitve Inhibitor 1The structure is similar to substrate and the binding site is just the active siteCompetitve Inhibitor 2The structure is not similar to substrate and the binding site is other than the active siteA classic example - Succinate dehydrogenaseAnother exampl

19、e- Dihydrofolate reductaseOne more example HIV proteaseHIV protease needs to bind and cleave polypeptide during life cyclevSVmaxKm1/S1/v1/Vmax-1/KmSlope=Km/Vmaxk1K-1k2KIKm increasesVmax unchanged+inhibitorKm(1+I/KI)-1/(Km(1+I/KI)Slope= Km(1+I/KI)/VmaxKinetics for Competitive Inhibitionk1k-1k2KIKm un

20、changedVmax decreasesKI+inhibitor1/S1/v1/Vmax-1/KmSlope=Km/Vmax(1+I/KI)/VmaxSlope= Km(1+I/KI)/VmaxvSVmaxKmKmVmax/(1+I/KI)Kinetics for petitive Inhibitionk1k-1k2Km decreasesVmax decreasesSlope unchanged+inhibitorKI1/S1/v1/Vmax-1/KmSlope=Km/Vmax(1+I/KI)/VmaxSlope= Km/VmaxvSVmaxKmKm/(1+ I/KI)Vmax/(1+ I

21、/KI)- (1+I/KI)/KmKinetics for petitive InhibitionIrreversible InhibitorCombines with or destroys an essential functional group on the enzyme (e.g. forms covalent bonds)Inhibit enzymes irreversiblyKinetic behavior resembles petitive inhibitorFour different types:Group Specific Reagent: - inhibitor do

22、es not resemble substrateSubstrate Analogue: - inhibitor resembles substrateTransition-state Analogue: - inhibitor resembles transition stateSuicide Inhibitors: - inhibitor resembles substrate, turns dangerous after processed by enzyme Group Specific ReagentDoes not resemble substrateirreversibly in

23、activates enzyme by modifying an essential R group e.g. DIPF (potent nerve gas) blocks acetylcholinesterase, which is essential for proper neural transmissionactive site serineSubstrate AnalogueResemble substrateBinds at enzyme active siteThen irreversibly modifies (or binds to) to the active sitee.

24、g. TPCK forms covalent bond with the Histidine at the active site of Chymotrypsin (a protease secreted by the pancreas) An example of Substrate AnalogueTransition state analogsA compound that mimics a transition state exploits these binding interactions in ways that a substrate analog cannot, so it

25、can be a particularly effective inhibitor. Suicide InhibitorResemble substrateBinds at enzyme active site (not an inhibitor yet)Processed by enzyme via normal catalytic mechanism es a chemically active intermediates that modifies and inactivates the enzyme irreversiblyGood candidate for drug due to

26、minimal side effectPenicillin is a suicide inhibitorAllosteric enzyme, Allostery and CooperativityAllosteric enzymes are enzymes having allosteryThe terms allostery and cooperativity have been muddled.Allostery strictly refers to influence of activity by a distant site.Cooperativity indicates that t

27、he occupancy of one site in a multisubunit enzyme influences the binding on the others. This is a form of allostery, but is only one manifestation of a general phenomena.Unfortunately allostery had e almost exclusively associated with the behavior of multi-subunit enzymes.Kinetics of Allosteric Enzy

28、meAllostery: Websters DefinitionMain Entry: allosteric Pronunciation: a-lO-ster-ik, -stir-Function: adjectiveEtymology:all- + stericDate: 1962Meaning: of, relating to, or being a change in the shape and activity of a protein (as an enzyme) that results from combination with another substance at a po

29、int other than the chemically active site- allosterically /-i-k(&-)lE/ adverb- allostery /a-lO-ster-E, -stir-/ nounGeneral Properties of Allosteric EnzymesThe activities of allosteric enzymes are changed by metabolic inhibitors and activators.Allosteric modulators bind noncovalently to the enzymes t

30、hey regulate.With few exceptions, regulated enzymes are multisubunit proteinsAn allosterically regulated enzyme usually has at least one substrate for which the v0 versus S curve is sigmoidal rather than hyperbolicCompetitive inhibitors can activate allosteric enzymes at a very low concentrationAs c

31、ompared to Michaelis enzyme, allosteric enzyme are minoritiesKinetic Signature of Cooperativity in Allosteric EnzymesMultisubunit enzymes that exhibit cooperativity show a sigmoidal initial velocity curve in contrast to the hyperbolic curve for independent subunits.Kinetic Consequences of Allosteric

32、 Effectors on Cooperative EnzymesThis is the traditional view of feed-back inhibition and regulation in “allosteric” enzymes.Classic Examples of AllosteryHemoglobin (not an enzyme)This was the origin of the T and R statesAspartate transcarbamoylase.Example of feedback inhibitionThese are great examp

33、les, but there are many others.Models for Allosteric RegulationConcerted (conceptually simple and often effective)Sequential (probably correct but difficult to prove)The Concerted ModelAssumes 2 conformation states: R & T Binding of substrate induces all subunits to change to R state.No T-R hybrids.

34、Allows for + cooperativity only.+ cooperativityT stateR stateThe Sequential ModelThe binding of substrate switches conformation of only the subunit to which it is bound.Conformational change in one subunit may or the affinity of other subunits have for the substrate.Allows for + or - cooperativity.B

35、oth the sequential and concerted models account for allosteric regulation of cooperative enzymes Allosteric activators that alter the Km of an enzyme stabilize the high affinity state of the active site. Allosteric activators that alter the kcat of an enzyme stabilize the high activity state of the active site. Allosteric inhibitors that alter the Km of an enzyme stabilize the low affinity state of the active site. Allosteric inhibi