Traveling over a mountain pass is an analogy frequently used to describe the progress of a chemical reaction. Catalysts speed up the process.
FIGURE 6.1 Activation energy profiles. (a) The activation energy profile for a typical reaction. The reaction shown here is exergonic (energy-releasing). Note the difference between the activation energy ( G °‡) and the standard free energy of the reaction ( G °).
FIGURE 6.1 Activation energy profiles. (b) A comparison of activation energy profiles for catalyzed and uncatalyzed reactions. The activation energy of the catalyzed reaction is much less than that of the uncatalyzed reaction.
The possible isozymes of lactate dehydrogenase. The symbol M refers to the dehydrogenase form that predominates in skeletal muscle, and the symbol H refers to the form that predominates in heart (cardiac) muscle.
FIGURE 6.2 The effect of temperature on enzyme activity. The relative activity of an enzymatic reaction as a function of temperature. The decrease in activity above 50°C is due to thermal denaturation.
FIGURE 6.3 Two models for the binding of a substrate to an enzyme. (a) In the lock-and-key model, the shape of the substrate and the conformation of the active site are complementary to one another. (b) In the induced-fit model, the enzyme undergoes a conformational change upon binding to substrate. The shape of the active site becomes complementary to the shape of the substrate only after the substrate binds to the enzyme.
FIGURE 6.4 The activation energy profile of a reaction with strong binding of the substrate to the enzyme to form an enzyme–substrate complex.
FIGURE 6.9 Graphical determination of V max and K M from a plot of reaction velocity, V, against substrate concentration, [S]. V max is the constant rate reached when the enzyme is completely saturated with substrate, a value that frequently must be estimated from such a graph.
FIGURE 6.10 A Lineweaver–Burk double reciprocal plot of enzyme kinetics. The reciprocal of reaction velocity, 1/ V, is plotted against the reciprocal of the substrate concentration, 1/[S]. The slope of the line is K M/ V max, and the y intercept is 1/ V max. The x intercept is –1/ K M.
FIGURE 6.11 Modes of action of inhibitors. The distinction between competitive and noncompetitive inhibitors is that a competitive inhibitor prevents binding of the substrate to the enzyme, whereas a noncompetitive inhibitor does not. (a) An enzyme– substrate complex in the absence of inhibitor. (b) A competitive inhibitor binds to the active site; the substrate cannot bind. (c) A noncompetitive inhibitor binds at a site other than the active site. The substrate still binds, but the enzyme cannot catalyze the reaction because of the presence of the bound inhibitor.
FIGURE 6.11 Modes of action of inhibitors. The distinction between competitive and noncompetitive inhibitors is that a competitive inhibitor prevents binding of the substrate to the enzyme, whereas a noncompetitive inhibitor does not. (b) A competitive inhibitor binds to the active site; the substrate cannot bind.
FIGURE 6.11 Modes of action of inhibitors. The distinction between competitive and noncompetitive inhibitors is that a competitive inhibitor prevents binding of the substrate to the enzyme, whereas a noncompetitive inhibitor does not. (c) A noncompetitive inhibitor binds at a site other than the active site. The substrate still binds, but the enzyme cannot catalyze the reaction because of the presence of the bound inhibitor.
FIGURE 6.12 A Lineweaver–Burk double-reciprocal plot of enzyme kinetics for competitive inhibition.
FIGURE 6.13 A Lineweaver–Burk plot of enzyme kinetics for noncompetitive inhibition.
Active site of VX-478 complexed with HIV-1 protease.
Transcript of "Ch02"
The Behavior of Proteins: Enzymes Chapter 02 : Enzyme kinetics 2.0 Factors affecting rate of reaction 2.1 Michelis-Menten & Lineweaver-Burke 2.2 Enzyme inhibition & regulation
Recall…• Activation energy? - the energy that used to initiate the reaction, where this energy is needed to break the chemical bonding so that the reaction can occur. - this energy is low with the usage of enzyme; do not influence the product (P), path of reaction and final concentration of molecules. proteins• Catalysis? - speed up the reaction; the catalysts that serve this function called enzymes. specific
Enzyme characteristics Protein Catalysts Specific reaction Reacts at optimal pH and temperature Regulate/control the metabolism processes Need in a low amount Reversible reaction Reaction may be inhibited by inhibitor
Factors affecting rate of reactioni. Enzyme concentration [E] – with a constant [S], the rate of reaction increased with the increasing [E]ii. Substrate concentration [S] – the rate of reaction increased until the amount of S = Eiii. pH – depends on functional group (R); -COOH or -NH2iv. Effect of temperature – increment of temperature will increase the rate of reactionv. Effect of inhibitor – chemical substance that binds on the active site/other site on the enzyme (allosteric site) → competitive and non-competitive inhibition
• The Lock-and-key model: high • The induced-fit model: the binding degree of similarity between the of the substrate induces the shape of substrate and the conformational change in the geometry of the binding site on enzyme. the enzyme. • The binding site has a different 3-• The substrate binds to a site D shape before the substrate is whose shape compliments to its bound. own. • The shape of the active site• eg. Like a key in lock or the becomes complementary to the correct piece in jigsaw puzzle. shape of the substrate only after• Weakness? the substrate binds to the enzyme. • Mimics the transition state.
Michaelis-Menten model• Devised in 1913 by Leonor Michelis and Maud Menten.• Basic model for nonallosteric enzyme.• The main feature of this model for enzymatic reaction is the formation of an E-S complex.• The [E-S] is low but remains unchanged to any appreciable extent over the course of the reaction.• The S → P; released from the E.• The E is regenerated at the end of the reaction. k1 k2 E + S ↔ ES → E + P k-1
• The rate (velocity) of an enzymatic reaction depends on the [S].• Fig. 6-8 shows the rate and the observed kinetics of an enzymatic reaction.• In lower region of the curve (at low level of S) – V0 depends on S.• In upper portion of the curve (at higher levels of S), the reaction is zero.• At infinite [S], the reaction would proceed at its max velocity (Vmax)
Vmax [ S ]• The [S] at which the V = reaction proceeds at KM +[S ] one-half its Vmax has a special significance.• It’s given the symbol KM (Michaelis constant) which considered an inverse measure of the affinity of the E for the S.• The lower the KM, the higher the affinity.• The Vmax for the E can be estimated from the graph. Thus, the value of KM also can be estimated from the Fig. 6-9, p.142
Vmax [S ]V = K M +[S ]• When experimental conditions are adjusted so that [S] = KM, Vmax [ S ] Vmax V = V = [S ] +[S ] and 2Note : Michaelis-Menten model is the simplest enzyme equation, where it’s considered the reaction of one single S to a single P. : the term KM only appropriate for E that exhibit a hyperbolic curve of V vs [S].
1 K +[ S ] = MV Vmax [ S ] Linearizing the1 = KM + [S ] Michaelis-MentenV Vmax [ S ] Vmax [ S ]1 K 1 = M × + 1 EquationV Vmax S Vmax • The curve that describe the rate of nonallosteric enzymatic reaction is hyperbolic. • It is considerably easier to work with straight line than a curve. • The equation for a hyperbola transformed into an equation for a straight line by taking the reciprocal of both sides: Lineweaver-Burk double reciprocal plot Fig. 6-10, p.143
Significance of KM and Vmax• When V = Vmax / 2, then KM = [S] → interpret that KM is equals the concentration of S at which 50% of the enzyme’s active sites are occupied by S.• Another interpretation of KM relies on the assumptions of the original Michaelis-Menten model of enzyme kinetics.• The KM is a measure of how tightly the S is bound to the E. KM >>, the less tightly the S bound to the E. Illustrate the• Vmax is related to the turnover number of an E, a quantity equal of the catalytic efficiency to constant,k2. ( Vmax / [ET]) = turnover number = kcat or kp enzymatic catalysis - no. of moles of S that react to form P/mole E/unit time.
How Do Enzymatics Reactions Respond to Inhibitors?Inhibitor – a substance that interferes with theaction of an enzyme and slows the rate of areaction.2 ways in which inhibitors can affect anenzymatic reaction:i.A reversible inhibitorii.An irreversible inhibitorThere 2 major classes of reversible inhibitorswhich can be distinguished on the basis ofthe sites on the E to which they bind:i.Competitive inhibitionii.Noncompetitive inhibition Fig. 6-11, p.146
Kinetics of competitive inhibitionIn the presence of competitive inhibitor, the equation for an enzymatic Important: substrate or inhibitor can bind thereaction becomes enzyme, not both. Because both are vying for the EI + I E ↔ ES → E + P ↔ +S same location, sufficiently high substrate will “outcompete” the inhibitor. This is why the VmaxThe dissociation constant for the E-I complex can be written: does not change. EI ↔ E + I KI = [E] [I] / [EI] 1 KM [I ] 1 1 = (1 + )× + V Vmax K I [ S ] Vmax y = m× x + b Fig. 6-12, p.148
Kinetics of noncompetitive inhibitionIn the presence of noncompetitive inhibitor, the reaction pathway hasbecome more complicated+S E ↔ ES → E + P +I ↕ ↕ +I +S EI ↔ ESI•The value of Vmax decreases, but KM remains the same; the inhibitordoesn’t interfere with the binding of S to the active site. 1 KM [I ] 1 1 [I ] = (1 + )× + (1 + ) Fig. 6-13, p.149
Kinetics of uncompetitive inhibition•The inhibitor can bind to the ES complex but not to free E.•The Vmax decreases and KM decreases as well.•Once the uncompetitive inhibitor biond to the complex, it willremain there. The enzymes loss their biology function →reaction STOP.•e.g. drugs, heavy metal (Boron), iodoacetic acid
• Practice session Sucrose is hydrolyzed to glucose and fructose. The reaction is catalyzed by the enzyme invertase. Using the following data, by the Lineweaver-Burk method, whether the inhibition of this reaction by 2 M is competitive or noncompetitive. [Sucrose] V, no inhibitor V, Inhibitor (mol L-1) present 0.0292 0.182 0.083 0.0584 0.265 0.119 0.0876 0.311 0.154 0.117 0.330 0.167 0.175 0.372 0.192 p.151a
Enzyme inhibition inthe treatment of AIDS –important target is HIVprotease that essentialto the production ofnew virus particles ininfected cells.Treatment is mosteffective whencombination of drugtherapies is used andHIV protease inhibitorsplay an important role.
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