第1页：Chapter 8 Enzymes Kinetics
Chapter 8 –Enzymes Kinetics
第2页：Introduction to Enzymes
- Introduction to Enzymes
- Thousands of biochemical reactions proceed at any given instant within living cells. These reactions are catalyzed by enzymes;
- Enzymes are mostly proteins. But two important enzymes are most certainly to be RNA (ribozymes). One is the ribosome (peptidyl transfer) and the other is the splicesome (splicing of intron);
- Enzymes are the agents of metabolic function. Enzymes play key functions in controlling rate of reaction, coupling reactions, and sensing the momentary metabolic needs of the cell.
第3页：Enzymes—Catalytic
8.1 •Enzymes—Catalytic Power, Specificity, and RegulationEnzymes are characterized by three distinctive features: catalytic power, specificity,regulation.
第4页：Enzymes are
Enzymes are incredibly efficientCatalytic Power
第5页：Enzyme Specificity
Enzyme SpecificityThe selective qualities of an enzyme are collectively recognized as its specificity.Intimate interaction between an enzyme and its substrates occurs through molecular recognition based on structural complementarity; such mutual recognition is the basis of specificity. The specific site on the enzyme where substrate binds and catalysis occurs is called the active site.
第6页：of Proteases Trypsin
Enzyme Specificity of ProteasesTrypsinThrombin
第7页：Regulation
RegulationRegulationof enzyme activity is achieved in a variety of ways, ranging from controls over the amount of enzyme protein produced by the cell to more rapid, reversible interactions of the enzyme with metabolic inhibitors and activators. Next chapter is devoted to discussions of enzyme regulation. Because most enzymes are proteins, we can anticipate that the functional attributes of enzymes are due to the remarkable versatility found in protein structures.
第8页：Enzyme
Enzyme ClassificationNMP kinase: ATP + NMP ADP + NDPE.C. 2.7.4.4
第9页：Enzyme co-factors
Enzyme co-factorsApoenzyme + cofactor = holoenzyme
第10页：Cofactor(Coenzyme
Cofactor(Coenzyme and Prosthetic Group)In most cases, a coenzyme is firmly associated with its enzyme, perhaps even by covalent bonds, and it is difficult to separate the two. Such tightly bound coenzymes are referred to as prosthetic groupsof the enzyme. The catalytically active complex of protein and prosthetic group is called the holoenzyme.The protein without the prosthetic group is called the apoenzyme;it is catalytically inactive.
第11页：Enzymes can
Enzymes can transform energy as well as molecules
第12页：Introduction to
- 8.2 Introduction to Enzyme Kinetics
- Kineticsconcerns with the rates of chemical reaction. Enzyme kineticsaddresses the biological roles of enzymatic catalysts and quantify the remarkable function of biological 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. Thus the science of pharmacology relies on such information
第13页：Kinetics of
8.3 Kinetics of Enzyme-Catalyzed ReactionThe Rate Law of Unimolecular reactions (review)•Consider a reaction of overall stoichiometry, The rate, or velocity, vof this reaction is the amount of P formed or the amount of A consumed per unit time. Thus:Rate law states that: Where kis rate constant. vis a function of [A] to the first power, or the first order. kis called first order constant.
第14页：The Rate Law of
The Rate Law of Biomolecular Reactions (review)•Consider a reaction of overall stoichiometry, The rate, or velocity, vof this reaction is the amount of P or Q formed or the amount of A or B consumed per unit time. Thus:Rate law states that: Where kis rate constant. vis a function of [A][B], or second order. kis the second order rate constant.
第15页：Rate Constant and
Rate Constant and Free Energy of ActivationDG‡ (noncatalyzed)DG‡ (catalyzed)DGPAX‡Arrhenius Equation:Enzymes do not alter the potential of a chemical reaction (i.e., Gibbs freeenergy). They acceleratethe rateof reaction. In anotherword, they lowerthe activation energy.Enzyme can accelerate the rate of a reaction by as much as 1016.
第16页：Enzyme Kinetics
- Enzyme Kinetics
- The 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?v[A]v[A]
第17页：The Mechaelis-Menten
- The Mechaelis-Menten EquationYou need to know how this is derived
- This 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 the previous slide.
- Mechaelis-Menten equation is derived based on the following three conditions:1)State steady assumption;2)Initial velocity assumption;3)Rate law.k1K-1k2K-2
第18页：Steady State
- Steady State Assumption
- Steady state is defined as the state during which the enzyme-substrate complex, [ES], remains constant, or
- Pre-steady state is the state during which [ES] builds up, usually very fast;
- MM equation concerns the reaction rate that is measured only when the steady state has reached.k1k2K-2
第19页：Initial Velocity
- Initial Velocity Assumption
- In the beginning of the reaction, there is very little product, or [P] is small. So the amount of [ES] contributed 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-2
第20页：Rate Law in Enzyme
- Rate Law in Enzyme Catalyzed Reactions
- Rate law still applies in enzyme catalyzed reactions.
- The forward velocity, or rate, vfis,
- The reverse velocity or rate, or the rate of disappearance vdis,
- At steady state, there is no accumulation of [ES], thus:k1K-1k2
第21页：Derivation of
- Derivation of Michaelis-Menten Equation
- We need one more condition, that is, the total enzyme concentration, [Et] is the sum of that of enzyme-substrate 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 = k2[ES]. So:Lecture 22. Enzyme And Catalysis21
第22页：Notes on the MM
- Notes on the MM Equations
- The rate of production formation can usually be measured experimentally by monitoring the progress curve of production formation.
- The maximum rate can be reached at saturating substrate concentration, or when [S]So MM equation can be re-written as:Enzyme-catalyzed rate is saturated
第23页：Understanding Km
- Understanding Km
- Km is a constant derived from rate constants
- Kmis, under true Michaelis-Menten conditions, an estimate of the dissociation constant of E from S, becauseat equilibrium, Reversible reaction, dissociation constant isSo small Kmmeans tight substrate binding; high Kmmeans weak substrate binding.
- Kmequals to the substrate concentration at which v=1/2vmaxk1k-1
第24页：Understanding vmax
- Understanding vmax
- Vmaxis asymptotically approached as substrate is increased, but never reached in reality, because this requires that ALL enzymes are bound with substrates (think about chemical equilibrium);
- Turnover number, kcat, is defined asIt describes number of substrates converted into products per enzyme, per unit time. When the enzyme is saturated with substrate, or [S]>>[Et],k1k-1
第25页：Orders of the MM图示
第26页：Orders of the MM
- Orders of the MM Equation (2nd, 1st, 0thorders)
- When substrate concentration is low, it is first order in [S], or
- Under the same low substrate concentration, [Et] [E], so it is second order in [E] and [S] and kcat/Kmis called apparent second-orderrate constant:
- When substrate concentration is high, the MM equation is 0thorder in [S]:
第27页：The Catalytic
- The Catalytic Efficiency, kcat/Km
- kcat/Kmis an estimate of "how perfect" the enzyme is
- The upper limit for kcat/Km is the diffusion limit -the rate at which E and S diffuse together, because:
第28页：Linear Plots Can示意图
第29页：Linear Plots Can Be
- Linear Plots Can Be Derived from the Michaelis -Menten Equation(Be familiar with these plots)
- The MM equationcan be plotted as
- Where Kmcorresponds to the horizontal axis when the vertical axis is half way of Vmaxvalue.[S]vvmaxKm
第30页：Lineweaver-Burk Plot
- Lineweaver-Burk Plot of MM Equation (Be familiar with these plots)
- More conveniently, the MM equation can be plotted as a linear plot by rearranging the expression above to:1/[S]1/v1/vmax-1/KmSlope=Km/vmax
- Lineweaver-Burk, or double reciprocal Plot
第31页：Hanes-Wolff plot(Be
- Hanes-Wolff plot (Be familiar with these plots)
- More conveniently, the MM equation can be plotted as a linear plot by rearranging the expression above to:[S][S]/vKm/vmax-KmSlope=1/vmax
- Hanes-Wolff Plot
第32页：Effect of p H on
Effect of pH on Enzymatic ActivityEnzyme-substrate recognition and the catalytic events that ensue are greatly dependent on pH. An enzyme possesses an array of ionizable side chains and prosthetic groups that not only determine its secondary and tertiary structure but may also be intimately involved in its active site. Further, the substrate itself often has ionizing groups, and one or another of the ionic forms may preferentially interact with the enzyme.
第33页：Effect of
Effect of Temperature on Enzymatic ActivityMost enzymatic reactions double in rate for every 10°C rise in temperature (that is, Q10= 2, where Q10 is defined as the ratio of activities at two temperatures 10°apart) as long as the enzyme is stable and fully active.Not all enzymes are quite so thermally labile. For example, the enzymes of thermophilic bacteria (thermophilic = ”heat-loving”) found in geothermal springs retain full activity at temperatures in excess of 85°C
第34页：Enzyme Inhibition
- 8.4 Enzyme Inhibition
- Enzyme can be inhibited by inhibitors. 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 enzymes through non-covalent interactions. Reversible inhibitors include three kinds:–Competitive inhibitors;–Non-competitive inhibitors;–Un-competitive inhibitors
- Irreversible inhibitorsassociate with enzymes through covalent interactions. Thus the consequences of irreversible inhibitors is to decrease in the concentration of active enzymes.
第35页：Enzyme inhibition
Enzyme inhibition Competitive vs. noncompetitive
第36页：Competitive
Competitive inhibitors are often structurally similar to substrates for the enzyme
第37页：Succinate
Succinate Dehydrogenase—A Classic Example of Competitive Inhibition
第38页：Competitive
Competitive Inhibitorsv[S]vmaxKm1/[S]1/v1/vmax-1/KmSlope=Km/vmaxk1K-1k2KIKmincreasesvmaxunchanged+inhibitorKm(1+[I]/KI)-1/(Km(1+[I]/KI))Slope= Km(1+[I]/KI)/vmax
第39页：Noncompetitive(Pure)
Noncompetitive (Pure) Inhibitorsk1k-1k2KIKmunchangedvmaxdecreasesKI’+inhibitor1/[S]1/v1/vmax-1/KmSlope=Km/vmax(1+[I]/KI)/VmaxSlope= Km(1+[I]/KI)/vmaxv[S]vmaxKmKmVmax/(1+[I]/KI)
第40页：Uncompetitive结构图
第41页：Uncompetitive
Uncompetitive Inhibitorsk1k-1k2KmdecreasesvmaxdecreasesSlope unchanged+inhibitorKI’1/[S]1/v1/vmax-1/KmSlope=Km/vmax(1+ KI/[I])/VmaxSlope= Km/vmaxv[S]vmaxKmKm/(1+ KI/[I])Vmax/(1+KI/[I])-(1+ KI/[I])/Km
第42页：Mixed Inhibitions(Be
Mixed Inhibitions (Be familiar with these plots)+inhibitor1/[S]1/v1/vmax-1/KmSlope=Km/vmax+inhibitor1/[S]1/v1/vmax-1/KmSlope=Km/vmax
第43页：Summary of Classes
- Summary of Classes of Inhibitors
- Competitive inhibition -inhibitor (I) binds only to E, not to ES
- Noncompetitive inhibition -inhibitor (I) binds either to E and/or to ES
- Uncompetitive inhibition -inhibitor (I) binds only to ES, not to E. This is a hypothetical case that has never been documented for a real enzyme, but which makes a usefulcontrast to competitive inhibition.
- Mixed inhibition-when the dissociation constants of (I) to E and ES are different. The inhibition is mixed.
第44页：Irreversible
- Irreversible Inhibition
- Irreversible inhibitors combine irreversibly with enzymes.
- Several drugs in current medical use are irreversible inhibitors of enzymes, or mechanism-based enzyme inactivators. For instance, penicllin exerts its effects by covalently reacting with an essential serine residue in the active site of glycoprotein peptidase, an enzyme that acts to cross-link the peptidyglycan chains during synthesis of bacterial cell walls.
第45页：inhibitors Modify enzymes
Irreversible inhibitorsModify enzymes by formation of new covalent bonds
第46页：Affinity labels
Affinity labelsSubstrates can be modified to generate specific covalent labeling reagents
第47页：Suicide inhibitors
Suicide inhibitorsReactive intermediate generated by enzyme inactivates the enzyme
第48页：Penicillin inhibits
Penicillin inhibits bacterial wall synthesisGlycopeptide transpeptidase
第49页：Structural
Structural similarity of penicillin and the D-Ala-D-Ala peptide
第50页：Transition State
Transition State Analogs are Potent and Specific InhibitorsProline racemase
第51页：RNA and Antibody
- 8.6
- RNA and Antibody Molecules as Enzymes: Ribozymes and Abzymes
- Ribozymes
- These catalytic RNAs, or ribozymes,satisfy several enzymatic criteria: 1.They are substrate-specific, 2.they enhance the reaction rate, 3.and they emerge from the reaction unchanged.
第52页：RNA splicing in
RNA splicing in TetrahymenarRNA maturation: (a) the guanosine-mediated reaction involved in the autocatalytic excision of the TetrahymenarRNA intron, and (b) the overall splicing process. The cyclized intron is formed via nucleophilic attack of the 3'-OH on the phosphodiester bond that is 15 nucleotides from the 5'-GA end of the spliced-out intron. Cyclization frees a linear 15-mer with a 5'-GA end.
第53页：Protein-Free 50 S
Protein-Free 50S Ribosomal Subunits Catalyze Peptide Bond Formation In VitroPerhaps the most significant case of catalysis by RNA occurs in protein synthesis. peptidyl transferase reaction,which is the reaction of peptide bond formation during protein synthesis
第54页：Several features of
Several features of these “RNA enzymes,” or ribozymes,lead to the realization that their biological efficiency does not challenge that achieved by proteins. First, RNA enzymes often do not fulfill the criterion of catalysis in vivobecause they act only once in intramolecular events such as self-splicing. Second, the catalytic rates achieved by RNA enzymes in vivoand in vitroare significantly enhanced by the participation of protein subunits. Nevertheless, the fact that RNA can catalyze certain reactions is experimental support for the idea that a primordial world dominated by RNA molecules existed before the evolution of DNA and proteins.
第55页：Catalytic antibodies
Catalytic antibodiesAntibodies that recognize a transition state will catalyze a specific reaction -ferrochelatase
第56页：Chapter 8-Review
- Chapter 8 -Review
- Enzymes possess catalytic power and specificity
- Enzymes lower free energy of activation but don’t change the equilibrium
- The Michaelis-Menten model explains many of the properties of enzymes
- Enzyme inhibitors can act in several ways
