Unit 3: Enzyme & Catalysis Kinetics - BIOC2200 PDF

Unit 3: Enzyme & Catalysis BIOC/BIOL 2200 Image credit: “REMOVE” created by Irina Bezsonova, CC-BY- 4.0 PDB structure used in this illustration: 1NBF, 3HP7...

Unit 3: Enzyme & Catalysis BIOC/BIOL 2200 Image credit: “REMOVE” created by Irina Bezsonova, CC-BY- 4.0 PDB structure used in this illustration: 1NBF, 3HP7 Dr. Pettit  Lesson 3.2 Enzyme Kinetics Learning Goals Through this lesson, you will learn: the importance of initial rates as applied to enzyme kinetics and how substrate concentration affects enzyme kinetics. to understand the Michaelis-Menten Equation and the meaning of its components how to use kinetic parameters to describe how enzymes behave the advantages and disadvantages of direct and Lineweaver-Burk plots of kinetic data how to construct and interpret direct and double reciprocal plots using raw kinetic data to determine kinetic parameters (Vmax, Km, and kcat) from data Let’s consider the degradation of serotonin There is a family of enzymes that catalyze the oxidation of monoamines such as the neurotransmitter serotonin. These enzymes use oxygen to remove an amine group. Enzyme Specificity Remember - enzymes are chiral since they are made up of only L amino acids. Therefore, they are stereospecific in the substrates they bind and the reactions they catalyze. e.g. lactate dehydrogenase can only recognize L – lactate (not D-lactate)  Lock and Key vs Induced Fit substrate enzyme Source - Figure 4.6 – Biochemistry Free for All Let’s consider how the neurotransmitter serotonin might interact with the active site of its degradative enzyme (MAO): H NH3 + O N H Enzymatic Reaction E+S ⇌ ES ⇌ ES* ⇌ EP ⇌ E+P * This represents the reaction occurring ~ we assume the simplest case = ES proceeds directly to E+P ~ E+S ⇌ ES ⇌ E+P Image Source – OpenStax Concepts of Biology Figure 4.8 Enzyme Kinetics → the study of enzyme reaction rates Why care about enzyme kinetics? Considering how rates change as conditions change tells us about the path followed by the reactants and the reaction mechanism facilitated by the enzyme Kinetics combined with the study of an enzyme’s structure and catalytic mechanism give us powerful insight into an enzyme’s biological function and potential implications for its use in various biotechnological, and/or therapeutic applications  For the enzyme catalyzed reaction: E+S ⇌ ES ⇌ E+P the progress of the reaction can also be depicted as a function of the change in concentration of each of its component parts. Fundamentals of Biochemistry 5th Ed Fig 12-2 Each elementary reaction (simple molecular reaction) that make up this enzyme catayzed reaction is described by a rate constant (k1, k-1, k2) k1 k2 E+S ⇌k ES → E+P-1 In this reaction scheme, the formation of product is a first order (unimolecular) reaction where the reaction rate (velocity, v) is: 𝑑[𝑃] v= = k2[ES] 𝑑𝑡 The rate of ES production is a function of both its appearance and disapearance 𝑑[𝐸𝑆] = k1[E][S] - k-1[ES] - k2[ES] 𝑑𝑡 This equation cannot be integrated without two imporatant assumptions Enzyme kinetics often follow the Michaelis-Menten model Researcher Spotlight Dr. Maud Menten Visit the Canadian Medical Hall of Fame to read more about this distinguished Canadian scientist and Leonor Michaelis (1875-1949) and Maud Menten (1879-1960) physician. Enzyme catalyzed reaction We cannot actually measure all of these progress. values. E+S ⇌ ES ⇌ E+P We assume: P is not converted back to S (irreversible) At [S], ES achieves steady state with k1 and k-1 in equilibrium k1 k2 E+S ⇌k ES → E+P -1 Fundamentals of Biochemistry 5th Ed Fig 12-2 How we Follow Reactions How we Follow Reactions – Continuous Assay Amount of product To meet the steady state requirement this must be measured within the first Initial velocity (Vo) must be measured during 10% of the reaction this initial linear burst phase of the reaction occurring when [S] > [E] Time (sec) Initial Velocity (V0) - Measured as [Product]/Time Substrate Concentration (Molarity) Enzyme Kinetics often follow the Michaelis-Menten model Remember, a reaction must have sufficient activation enegry to achieve its transition state (ES). E+S ⇌ ES ⇌ E+P With sufficient activation energy and a high substrate concentration ([S]), the enzyme (E) will become saturated ES → E+P is the rate limiting and the reaction will be insensitive to further [S] *For simiplicity we assume this reaction is not reversible Source - Figure 4.3 – Biochemistry Free for All KM is the Michaelis constant for an k1 k2 enzyme catalyzed reaction. E+S ⇌ ES → E+P k-1 k−1+ k2 Where KM = k1 KM = is the substrate concentration([S]) where the initial reaction velocity is at half of its maximum KM is a measure of an enzyme’s affinity for a given substrate Enzyme Km (mmol l-1) High Km = Low Affinity Carbonic anhydrase 26 Low Km = High Affinity Chymotrypsin 15 Remember: Ribonuclease 8 KM is unique for each enzyme-substrate pair Tyrosyl-tRNA synthase 0.9 An enzyme that can act on more than one Pepsin 0.3 substrate will have a different KM for each one If more than one enzyme works on the same substrate, each will have a different KM KM is also influenced by the physicochemical properties (e.g. - temp and pH) of the environment  k1 k2 E+S ⇌k ES → E+P -1 Michaelis-Menten Equation Vmax [𝑺] vo = 𝑲𝑴+[𝑺] I will not ask you to derive this equation but understanding how we arrived at it is exceedingly helpful to being able to use it and work with the related data. An enzyme-catalyzed reaction has a KM of 10 mM and a Vmax of 12 nMs-1. What is the reaction velocity when the substrate concentration is 2 mM? KM is a measure of an enzyme’s affinity for a given substrate If two enzymes, in different pathways, compete for the same substrate, knowing the values of KM and Vmax for both enzymes permits prediction of the metabolic fate of the substrate and the relative amount that will flow through each pathway under various conditions. We can determine kinetic parameters (Vmax and by extension KM) from a Michaelis-Menten plot (Vo vs [S]) since at very high [S] the curve will approach Vmax asymptotically In reality it is very difficult to assess Vmax directly from the plot even at very high [S], Vmax will often be underestimated A better option is to use the double reciprocal plot  Lineweaver-Burk Taking the reciprocal of the Michaelis-Menten equation gives: (double reciprocal plot) 1 𝐾𝑀 1 1 = + 𝑣𝑜 𝑉𝑚𝑎𝑥 [𝑆] 𝑉𝑚𝑎𝑥 1 This is a linear equation where is 𝑣𝑜 1 plotted against [𝑆] X and Y intercepts can be used to determine KM and Vmax respectively Potential limitations: Lineweaver-Burk Clustering of values even when a good range of [S] is assessed (double reciprocal plot) At small [S], error is magnified and can affect KM and Vmax values calculated significantly Remember - reaction rate (V) varies with [E] It is desirable to have a measure of velocity that is independent of enzyme concentration. Each enzyme also has a catalytic constant or turnover number, this is the number of reactions that each active site catalyzes per unit time Vmax kcat = 𝐸 𝑇 kcat/KM is a rate constant reporting how often an enzyme and substrate encounter one another in solution kcat/KM = catalytic efficiency Enzyme kcat/Km (s-1 M-1) Acetylcholinesterase 1.6 x 108 The upper limit to this efficiency (108 Carbonic anhydrase 8.3 x 107 Catalase 4.0 x 107 to 109 M-1S-1) is determined by the Crotonase 2.8 x 108 diffusion-controlled limit. Fumerase 1.6 x 108 At this upper limit, almost every Triose phosphate isomerase 2.4 x 108 encounter between an enzyme and Β-lactamase 1.0 x 108 substrate molecule results in the Superoxide dismutase 7 x 109 production of a product Not all reactions are simple! Thus far we have been considering the simple, single substrate reaction that obeys the Michaelis-Menten model: E+S ⇌ ES ⇌ E+P However, reactions with multiple substrates that yeild multiple products are FAR more common. Bisubstrate reactions (shown below) represent ~60% of biochemical reactions. Most often these are: Group transfer reactions Oxidation-reduction reactions Caffeine is catabolized by cytochrome P450 Figure 1, Kot M. and W. Daniel, Biochemical Pharmacology 76 (2008) 543–551 Contribution of various P450 isoforms to caffeine catabolism P450 Km Vmax Vmax/Km [E] is equal for each isoform 1A2 2.56 0.341 0.133 2A6 40.64 0.068 0.002 2B6 2.31 0.019 0.008 2C8 0.92 0.014 0.015 2C9 2.17 0.021 0.010 2C18 4.93 0.042 0.008 2C19 11.93 0.154 0.013 2D6 74.74 1.242 0.017 2E1 n.d. n.d n.d. 3A4 16.04 0.107 0.007 3A5 0.88 0.011 0.012 n.d.—not detected, Km [mM], Vmax [pmol/pmol P450 isoform/min] Data from Table 2, Kot M. and W. Daniel, Biochemical Pharmacology 76 (2008) 543–551 Learning Outcomes Having completed this lesson, you should be able to: calculate an initial rate from experimental data describe the effect of substrate concentration and product inhibition on the behavior of enzymes understand why enzymes saturate resulting in hyperbolic rate vs. substrate concentration plots apply the Michaelis Menten equation to partial data to solve for one of its components construct and interpret direct and double reciprocal plots using raw kinetic data estimate the kinetic parameters Vmax and Km from enzyme saturation data calculate the kinetic parameters (Vmax, Km, Kcat) from enzyme saturation data using direct fitting and Lineweaver-Burk methods compare and contrast enzymes based on their kinetic parameters