Biochemistry Department. Level 1 Lecture No : 3 Date : 1 / 10 / Enzymes kinetics

Transcription

1 Biochemistry Department Level 1 Lecture No : 3 Date : 1 / 10 / 2017 Enzymes kinetics 1

2 Intended Learning Outcomes By the end of this lecture, the student will be able to: 1.Understand what is meant by enzyme Kinetics 2.Define rate of reaction 3.Know Michaelis&Menten equation. 4.Define Michaelis constant Km & its importance (significance) 5.Identify Lineweaver- Burk plot& its significance. 6.Identify factors affect rate of reaction.

3 Enzyme Kinetics Is the study of rate of change of reactants to products by graphical expression Reaction velocity V or ( Rate of reaction) is: The number of substrate molecules converted to product per unit time. E + S <---> [ES] <---> E + P

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5 In 1913 Leonor Michaelis & Maud Menten: made a simple model of enzyme reaction. They made a mathematical correlation between [S] and the rate of reaction V that show a hyperbolic curve

6 hyperbolic curve V vs. [S] curve defines a rectangular hyperbola [hyperbolic curve] At low [S], V is directly proportional to [S]. At high [S], a Vmax is reached.

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8 Michaelis & Menten equation the M & M equation is : Vo = Vmax [S] Km + [S] Vo= initial reaction velocity [S]= substrate concentration V max= maximal velocity Km= the Michaelis Constant

9 V0 = initial velocity is the velocity measured when very little substrate has reacted. It is dependant on both substrate and enzyme concentration Vmax = maximal velocity The velocity obtained when all enzyme molecules are saturated with substrate

10 The Michaelis Constant Km Km: The Michaelis Constant is the substrate concentration, when enzyme velocity is equal to ½ V max. Half of enzyme molecules are saturated with the substrate. It is expressed as mole/liter.

11 Km (The Michaelis Constant) 1- It is constant for a particular enzyme under standard conditions. 2- It measures "realtive affinity" of an enzyme for its substrate. There is an inverse correlation between Km & enzyme Affinity Low Km High affinity of E to S High Km.low affinity of E to S

12 Glucokinase Hexokinase Km Km

13 3- It permits evaluation of inhibitor type. 4- It can be affected by many factors like ph & temperature.

14 How to determine Km? 1-from M & M curve: Not very accurate.

15 2-From Hans Lineweaver & Dean Burk plot By taking the reciprocal مقلوب of both sides of M & M equation Vo, S, Vmax Intercept on X axis = -1/Km Intercept on Y axis = 1/Vmax **slope = Km Vmax

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17 Linweaver-burk plot Hyperbolic curve Of M- M Both curves are very important for studying enzyme inhibition

18 Factors affecting reaction velocity 1- Temperature 2- ph 3- Substrate concentration

19 other factors: 4-Enzyme concentration: Increase enz. Conc. Will increase the rate of enz. Catalysed reaction. 5-Effect of coenzymes: Disscused before. 6-Effect of metals: before. Disscused

20 3-Substrate concentration: Increase in substrate concentration will increase the rate of enzyme activity till a certain level at which V-max is reached at which ALL THE ACTIVE SITES OF ENZYMES ARE SATURATED WITH THE SUBSTRATE With enz. Conc. Kept constant

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22 4-Enzyme concentration: ncrease enz. Conc. will increase the rate of enz. catalysed reaction.

23 Factors affecting reaction velocity 1-Temperature. The rate of any reaction increases as temperature rises within limits. Temperature coefficient= Q10; the velocity of enzyme doubles with a 10 c rise in temp.

24 The 'optimum' temperature for the enzyme is the temperature at which enzyme act by its full rate (37 c) i.e Vmax is reached. Bell shaped curve

25 At very high temperatures, the enzyme will completely denature (like egg white), becoming catalytically inactive.

26 When body temp. is lowered all enzymatic activities decrease (important in open heart surgery & in organ transplant Organ cryopreservation) to reduce the enzyme activity Preventing tissues damage.

27 2-pH. Each Enzyme has optimum ph at which it act by its full activity. Ex: pepsin (ph 1.5-2), salivary amylase (6.8) Bell shaped curve pancreatic amylase (7.5-8). Most enzymes have optimum ph (5-9).

28 Changes of ph above or below the optimum ph lead to denaturation of enzyme protein with loss of the catalytic activity due to change enzyme conformation.

29 Effect of optimum ph on enzyme activity: 1. Change the enzyme conformation so increasing substrate binding. 2. Make effect on charges on the active site of enzyme or on substrate, facilitate binding and catalysis.

30 MCQ Enzymes lower energy barrier by 1-change ph to be the optimum 2- raise the temperature 3- c formation of ES complex 4- make covalent bonds with the substrate

31 What happens to an enzyme when it denatures? A) The activation energy of the reaction is doubled. B) The activation energy of the reaction is lowered. C) the optimal conditions for temperature of the enzyme are doubled. D) c The shape of the enzyme molecule is changed.

32 In enzyme kinetics Vmax reflects: (A) The amount of an active enzyme (B) Substrate concentration (C) Half the substrate concentration (D) Enzyme substrate complex

33 At a substrate concentration that produces half maximal velocity: A- The substrate is half-saturated with the enzyme B- The enzyme is half saturated with the substrate C-The velocity is equal to Km D- the reaction is at equilibrium E- One-half of the substrate has changed to product

34 Reactions in living cells are distinguished from other chemical reactions by: A- proceeding at a fast rate B-being catalyzed C-proceeding in two directions D-having a negative free energy change

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