CHAPTER 3: ENZYMES Shuler, M. L. and Kargi. (2002). Bioprocess Engineering: Basic Concept. 2 nd Ed. Upper Saddle River, NJ: Prentice Hall PTR PTT203: BIOCHEMICAL ENGINEERING SEMESTER 1 (2014/2015) By: Puan Nurul Ain Harmiza 1
COURSE OUTCOME 1: Ability to DIFFERENTIATE types of enzymes and analyze its kinetics study and catalysis. By: Puan Nurul Ain Harmiza 2
ENZYMES: PART 1 OUTLINE: INTRODUCTION ENZYME REACTION [ENZYME-SUBSTRATE] INTERACTION MULTISUBSTRATE ENZYME-CATALYZED REACTION By: Puan Nurul Ain Harmiza 3
INTRODUCTION • Enzymes: – A re “active” proteins (>15k Da) that can catalyze (increase) the rate of biochemical reactions by breaking and making chemical bonds. – Are produced by living cells such as plant, animal and microorganism. – Enzymes are specific to their substrate. – The specificity are determined by the active site. By: Puan Nurul Ain Harmiza 4
• Substrates: – Are the reactants that are activated by the enzyme. By: Puan Nurul Ain Harmiza 5
ENZYME REACTION • Enzymes lower the activation energy of the reaction catalyzed by binding the substrate and forming an enzyme-substrate, [ES] complex. • Enzymes do not affect the free-energy change or the equilibrium constant. By: Puan Nurul Ain Harmiza 6
THE ACTION OF AN ENZYME FROM THE ACTIVATION-ENERGY POINT OF VIEW By: Puan Nurul Ain Harmiza 7
• The interaction between the enzyme and its substrate is usually by weak forces such as van der Waals forces and hydrogen bonding. • The substrate binds to a specific site on the enzyme known as the active site. • The substrate in a small molecule and fits into a certain region on the enzyme molecule which is a larger molecule. By: Puan Nurul Ain Harmiza 8
Example: Amylase By: Puan Nurul Ain Harmiza 9
ENZYME-SUBSTRATE INTERACTIONS (ES) COMPLEX MODES OF ACTIVITY: 1. LOCK-AND-KEY MODEL By: Puan Nurul Ain Harmiza 10
ENZYME-SUBSTRATE INTERACTIONS (ES) COMPLEX MODES OF ACTIVITY: 1. LOCK-AND-KEY MODEL 2. INDUCED-FIT MODEL By: Puan Nurul Ain Harmiza 11
In multi-substrate enzyme-catalyzed reactions, enzymes can hold substrates such as: • proximity effect : – that the reactive regions of substrates are close to each other and to the enzyme’s active site. • orientation effect : – enzymes may hold the substrate at certain positions and angles to improve the reaction rate. By: Puan Nurul Ain Harmiza 12
ENZYMES: PART 2 OUTLINE: ENZYME KINETICS By: Puan Nurul Ain Harmiza 13
INTRODUCTION • Kinetics of simple enzyme- catalyzed reactions are often referred to as Michaelis- Menten kinetics or saturation kinetics . By: Puan Nurul Ain Harmiza 14
• An enzyme solution has a fixed number of active sites to which substrate can bind. • At high substrate concentrations, all these sites may be occupied by substrates or the enzymes is saturated . • Saturation kinetics can be obtained from a simple reaction scheme that involves a reversible step for [ES] complex formation and a dissociation step of the [ES] complex. k 2 k 1 E S ES E P k -1 By: Puan Nurul Ain Harmiza 15
Two major approaches used in developing a rate expression for the enzyme-catalyzed reactions are: k 2 k 1 E S ES E P k -1 Rapid-equilibrium Quasi-steady-state approach approach [ ] V S V [ S ] m v m v K [ S ] [ ] K S m m By: Puan Nurul Ain Harmiza 16
Assignment 1 Please download and use the Assignment Sheet in Portal… Question 1 Derive the Michaelis-Menten equation through Rapid Equilibrium approach. Question 2 Derive the Michaelis-Menten equation through Quasi Steady State approach. By: Puan Nurul Ain Harmiza 17
EXPERIMENTALLY DETERMINING RATE PARAMETERS FOR MM TYPE KINETICS • The values Km and Vm can be determined by using batch reactor. INITIAL-RATE EXPERIMENTS Product [S] [S 0 ] Known concentrations [E 0 ] By: Puan Nurul Ain Harmiza 18
The K M is the substrate concentration where v o equals one-half V max Figure 14-8 Plot of the initial velocity v o of a simple Michaelis – Menten reaction versus the substrate concentration [S]. By: Puan Nurul Ain Harmiza 19
Double reciprocal plot (lineweaver-burk plot) • M-M equation is linearized double-reciprocal form: V [ S ] 1 1 K 1 m m v [ ] v V V [ S ] K S m m m • A plot of 1/ v vs. 1/[S] yields a linear line with a slope of K m /V m and y-axis intercept of 1/V m as in Figure 3.5 below: This plot gives good estimates on V m , but not necessarily on K m . • The error about the reciprocal of a data point is not symmetric bcoz • most experimental results crowded on one side of the graph. Data points at low substrate concentrations influence the slope and • intercept more than those at high substrate concentrations. By: Puan Nurul Ain Harmiza 20
Eadie-hofstee plot • Rearrangement of M-M equation into: V [ S ] v m m v v V K m K [ S ] [ S ] m • A plot of v vs. v/[S] results in a line of slope – K m and y-axis intercept of V m as in Figure 3.6 below: • This plot can be subject to large errors since both coordinates contain v, but there is less bias on data points at low [S]. By: Puan Nurul Ain Harmiza 21
Hanes-woolf plot • Rearrangement of M-M equation into: m [ S ] K 1 V [ S ] m [ S ] v v V V K [ S ] m m m • A plot of [S]/v vs. [S] results in a line of slope 1/V m and y- axis intercept of K m /V m as in Figure 3.7 below: [S]/v Slope=1/v max -K m K m /v max [ S ] • This plot is used to determine V m more accurately. By: Puan Nurul Ain Harmiza 22
Interpretation of Km and Vm • Km or K’m is intrinsic parameter while Vm is not. • Km is affected by the change in pH and temperature . • Vm is affected by the change in k2 and [E0] . By: Puan Nurul Ain Harmiza 23
ENZYMES: PART 3 OUTLINE: INHIBITED ENZYME KINETICS By: Puan Nurul Ain Harmiza 24
INTRODUCTION • Types of enzyme inhibitors: – Irreversible Inhibitors (Inactivators) – Reversible inhibitors • Competitive • Non-competitive (mixed) • Un-competitive By: Puan Nurul Ain Harmiza 25
Irreversible Enzyme Inhibition • Irreversible inhibitors associate with enzymes through covalent interactions. • The consequences of irreversible inhibitors is to decrease in the concentration of active enzymes (E T ) • Covalent modification of an enzyme may lead to loss of activity if: – an essential catalytic group is modified i.e. blocked – substrate binding is sterically hindered – the modification leads to some conformational distortion of the enzyme or mobility restraint By: Puan Nurul Ain Harmiza 26
Reversible Enzyme Inhibition • Many pharmaceuticals are enzyme inhibitors. • Reversible inhibitors associate with enzymes through non-covalent interactions. • Reversible inhibitors include three kinds: Competitive Uncompetitive Mixed inhibitors inhibitors inhibitors -bind to both E and -interfere with -bind to ES ES substrate binding complex By: Puan Nurul Ain Harmiza 27
Reversible Enzyme Inhibition Reversible inhibitors associate with enzymes through • non-covalent interactions. Reversible inhibitors include three kinds: 1. Competitive inhibitors 2. Mixed (Non-competitive) inhibitors 3. Un-competitive inhibitors By: Puan Nurul Ain Harmiza 28
Competitive Inhibition Inhibition constant E I K I EI By: Puan Nurul Ain Harmiza 29
Competitive Inhibition K M increases v max unchanged By: Puan Nurul Ain Harmiza 30
Competitive Inhibition: Lineweaver-Burk Plot Initial velocity in the presence of inhibitor V S max v o K S M I 1 K I MM equation: [ ] v S max v [ I ] [ S ] K ( 1 ) m K I By: Puan Nurul Ain Harmiza 31
Noncompetitive Inhibition Reversible Inhibitors The binding of the inhibitor will either alter the K M or V max or both. By: Puan Nurul Ain Harmiza 32
Noncompetitive (mixed) Inhibition: Lineweaver-Burk Plot By: Puan Nurul Ain Harmiza 33
Uncompetitive Inhibition • Uncompetitive Inhibition: Lineweaver-Burk Plot K m decreases v max decreases Slope unchanged k 1 k 2 E S ES E P k -1 + I K I ’ ESI By: Puan Nurul Ain Harmiza 34
Table 14-2 Effects of Inhibitors on the Parameters of the Michaelis – Menten Equation • Competitive inhibition Raises K M only (intercept in L-B plot) S and I compete for same binding site • Noncompetitive (mixed) inhibition Lowers V max (slope in L-B plot); may increase or decrease K M I binds at a site distinct from that at which the S binds • Uncompetitive inhibition Both V max & K M affected I binds to ES complex, but not free E Formation of an ESI complex which does not break down to products at a significant rate By: Puan Nurul Ain Harmiza 35
EFFECT OF PH AND TEMPERATURE By: Puan Nurul Ain Harmiza 36
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