Bài giảng Kỹ thuật phản ứng sinh học: Chương 2 Động học phản ứng enzyme, cung cấp cho người học những kiến thức như: Các khái niệm cơ bản; Xúc tác sinh học; Phương trình Michelis Menten; Động học phản ứng enzyme. Mời các bạn cùng tham khảo!
http://buihongquan.com Chương Động học phản ứng enzyme 2.1 Cá c khá i niệ m bả n 2.2 Xú c tá c sinh họ c 2.3 Phương trình Michelis Menten 2.4 Đọ ng họ c phả n ứng enzyme 12/23/2018 Bioreaction engineering 80 http://buihongquan.com QUIZ What is enzyme? What is the function of enzyme? What are the special characteristics of enzyme? What kind of binding energy involve for the formation of ES complexes (enzyme-substrate complex)? Define these terms: Active site Activation energy Cofactors Apoenzyme Holoenzyme Coenzyme Substrate 12/23/2018 Bioreaction engineering 81 http://buihongquan.com QUIZ • • • • • • • • 12/23/2018 What characteristic features define enzymes? Can the rate of an enzyme-catalyzed reaction be defined in a mathematical way? What equations define the kinetics of enzymecatalyzed reactions? What can be learned from the inhibition of enzyme activity? What is the kinetic behavior of enzymes catalyzing bimolecular reactions? How can enzymes be so specific? Are all enzymes proteins? Is it possible to design an enzyme to catalyze any desired reaction? Bioreaction engineering 82 http://buihongquan.com Role of Bioprocess Engineering exploit advances in biology to create new products design biochemical processes & operate plants develop energy resources Develop new, environmentally friendly, and safer processes to make the biochemical products that people depend on Work in research and development laboratories, creating polymeric materials with improved performance and durability Work in manufacturing, making vaccines and antibiotics Invent new ways to keep our food and water supplies safe 12/23/2018 Bioreaction engineering 83 http://buihongquan.com Bioprocess Engineer’s Task Minimize production of unwanted byproducts Separate the good (product) from the bad (byproducts) Recover the unused reactants Maximize profit, minimize energy consumption Minimize impact on the environment 12/23/2018 Bioreaction engineering 84 http://buihongquan.com OUTLINE Introduction Enzyme Structure Enzyme Function Enzyme Kinetics A)Michaelis –Menten Kinetics B) The Rapid Equilibrium Assumption C) The Quasi-Steady-State Assumption 12/23/2018 Bioreaction engineering 85 http://buihongquan.com Enzymes There are many chemical compounds in the living cell How they are manufactured and combined at sufficient reaction rates under relatively mild temperature and pressure? How does the cell select exactly which reactants will be combined and which molecule will be decomposed? Catalysis by ENZYME 12/23/2018 Bioreaction engineering 86 http://buihongquan.com Enzymes •Enzymes are biological catalysts that are protein molecules in nature- react in mild condition •They are produced by living cells (animal, plant, and microorganism) and are absolutely essential as catalysts in biochemical reactions •Almost every reaction in a cell requires the presence of a specific enzyme– related to its particular protein structure •A major function of enzymes in a living system is to catalyze the making and breaking of chemical bonds •Therefore, like any other catalysts, they increase the rate of reaction without themselves undergoing permanent chemical changes 12/23/2018 Bioreaction engineering 87 http://buihongquan.com Over 2000 enzymes have been identified Often named by adding the - ‘ase’ to the name of substrate acted upon, or the reaction catalyzed such as urease, alcohol dehydrogenase The majority of cellular reactions are catalyzed by enzymes 12/23/2018 Bioreaction engineering 88 http://buihongquan.com Some protein enzyme required a non-protein group for their activity Non protein group: Cofactors: metal ions, Mg, Zn, Mn, Fe Coenzyme: complex organic molecule, NAD, FAD, CoA Vitamins Catalyze biochemical reactions breaking, forming and rearranging bonds Catalytic function – very specific and effective (Specific because of conformational shape) Dictated by the enzyme active site Some active sites allow for multiple substrates 12/23/2018 Bioreaction engineering 89 http://buihongquan.com The Turnover Number Defines the Activity of One Enzyme Molecule A measure of catalytic activity kcat, the turnover 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 sec 12/23/2018 Bioreaction engineering 129 http://buihongquan.com The Turnover Number Defines the Activity of One Enzyme Molecule 12/23/2018 Bioreaction engineering 130 http://buihongquan.com The Ratio kcat/Km Defines the Catalytic Efficiency of an Enzyme The catalytic efficiency: kcat/Km An estimate of "how perfect" the enzyme is kcat/Km is an apparent second-order rate constant It measures how well 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 12/23/2018 Bioreaction engineering 131 http://buihongquan.com The Ratio kcat/Km Defines the Catalytic Efficiency of an Enzyme 12/23/2018 Bioreaction engineering 132 http://buihongquan.com Linear Plots Can Be Derived from the MichaelisMenten Equation Be able to derive these equations Lineweaver-Burk: Begin with v = Vmax[S]/(Km + [S]) and take the reciprocal of both sides Rearrange to obtain the Lineweaver-Burk equation: ỉ Km ỉ =ỗ + ữ ỗ ữ v ố Vmax ø è [S] ø Vmax A plot of 1/v versus 1/[S] should yield a straight line 12/23/2018 Bioreaction engineering 133 http://buihongquan.com Linear Plots Can Be Derived from the MichaelisMenten Equation The Lineweaver-Burk double-reciprocal plot 12/23/2018 Bioreaction engineering 134 http://buihongquan.com Linear Plots Can Be Derived from the MichaelisMenten Equation Hanes-Woolf: Begin with Lineweaver-Burk and multiply both sides by [S] to obtain: Km [S] ổ =ỗ [S] + ÷ v è Vmax ø Vmax Hanes-Woolf is best - why? Because Hanes-Woolf has smaller and more consistent errors across the plot 12/23/2018 Bioreaction engineering 135 http://buihongquan.com Linear Plots Can Be Derived from the Michaelis-Menten Equation A Hanes-Woolf plot of [S]/v versus [S] 12/23/2018 Bioreaction engineering 136 http://buihongquan.com Determination of Rate Parameters for Michaelis-Menten Type Kinetics Lineweaver-Burk plot Eadie- Hofstee plot Hanes-Woolf plot Batch kinetics 12/23/2018 Bioreaction engineering 137 Lineweaver-Burk Plot http://buihongquan.com From equation (Quasi-steady-state ), Vm [ S ] v Km [ S ] Double reciprocal plot slope Y-intercept Lineweaver-Burk plot gives good estimates on Vm but not necessarily on Km (error relates with substrate conc) 12/23/2018 Bioreaction engineering 138 Eadie–Hofstee Plot http://buihongquan.com Vm [ S ] From equation 6, v Km [ S ] Rearranged equation 6, plot v versus v/[S] gives a line of slope –Km and y-axis intercept of Vm Can be subject to large errors since both coordinates contain v, but less bias on points at low [S] 12/23/2018 Bioreaction engineering 139 http://buihongquan.com Hanes–Woolf plot Rearrangement of equation yields, intercept slope This plot is used to determine Vm more accurately 12/23/2018 Bioreaction engineering 140 http://buihongquan.com Batch kinetics Integration of equation and rearranged yields, [ So ] ln t [S] Km [ So ] [ S ] Vm t Km slope Y-intercept [ So ] ln t [S] [ So ] [ S ] t 12/23/2018 Bioreaction engineering 141 http://buihongquan.com Experiment Effect of Substrate Concentration on Enzyme Kinetics Study OBJECTIVES 1.To develop a suitable standard curve for enzyme assay 2.To analyze the effect of substrate concentration on the activity of enzyme 3.To determine Vmax and Km from the enzyme reaction using enzyme kinetics plots 12/23/2018 Bioreaction engineering 142 http://buihongquan.com COURSE OUTCOMES Ability to develop enzyme reactions based on its kinetics study and applied catalysis •INTRODUCTION The enzyme α-amylase can catalyze the hydrolysis of internal α -1,4-glycosidic bond present in starch with the production of reducing sugars In the study of substrate concentration on enzyme kinetics, the enzyme is kept constant where as the concentration of starch is taken in increasing order As the substrate concentration increases, the amount of products produced in every successive tube also increases This enzyme-substrate reaction can be determined by measuring the increase in reducing sugars using the 3,5-dinitrosalicylic acid reagent In an alkaline condition, the pale yellow coloured the 3,5-dinitrosalicylic acid undergo reduction to yield orange coloured 3-amino-5-nitrosalicylic acid The absorbance of resultant solutions is read at 540nm The intensity of colour depends on the concentration of reducing sugars produced 12/23/2018 Bioreaction engineering 143 ... of Substrate Concentration 12/ 23 /20 18 Bioreaction engineering 121 http://buihongquan.com 12/ 23 /20 18 Bioreaction engineering 122 http://buihongquan.com Quasi-Steady-State Assumption Briggs and... an analytical solution 12/ 23 /20 18 Bioreaction engineering 117 http://buihongquan.com Assumption (1) Rapid-Equilibrium Assumption (2) Quasi-Steady-State Assumption 12/ 23 /20 18 Bioreaction engineering... There is difference between Michaelis-Menten constant [K’m=k-1/k1] and Quasi-steady-state constant [K’m=k-1+k2/k1] 12/ 23 /20 18 Bioreaction engineering 125 http://buihongquan.com Understanding