CHAPTER 3: ENZYMES Shuler, M. L. and Kargi. (2002). Bioprocess - - PowerPoint PPT Presentation

CHAPTER 3: ENZYMES

By: Puan Nurul Ain Harmiza 1

PTT203: BIOCHEMICAL ENGINEERING SEMESTER 1 (2014/2015)

Shuler, M. L. and Kargi. (2002). Bioprocess Engineering: Basic Concept. 2nd

• Ed. Upper Saddle River, NJ: Prentice Hall PTR

COURSE OUTCOME 1:

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Ability to DIFFERENTIATE types of enzymes and analyze its kinetics study and catalysis.

ENZYMES: PART 1

OUTLINE: INTRODUCTION ENZYME REACTION [ENZYME-SUBSTRATE] INTERACTION MULTISUBSTRATE ENZYME-CATALYZED REACTION

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INTRODUCTION

• Enzymes:

– Are “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.

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• Substrates:

– Are the reactants that are activated by the enzyme.

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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
• r the equilibrium constant.

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THE ACTION OF AN ENZYME FROM THE ACTIVATION-ENERGY POINT OF VIEW

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• 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.

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Example: Amylase

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ENZYME-SUBSTRATE INTERACTIONS (ES) COMPLEX MODES OF ACTIVITY:

• 1. LOCK-AND-KEY MODEL

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ENZYME-SUBSTRATE INTERACTIONS (ES) COMPLEX MODES OF ACTIVITY:

• 1. LOCK-AND-KEY MODEL
• 2. INDUCED-FIT MODEL

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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.

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ENZYMES: PART 2

OUTLINE: ENZYME KINETICS

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INTRODUCTION

• Kinetics of

simple enzyme- catalyzed reactions are

• ften referred to

as Michaelis- Menten kinetics

• r saturation

kinetics.

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• 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.

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P E ES S E  

k1 k-1 k2

P E ES S E  

k1 k-1 k2

Rapid-equilibrium approach

] [ ] [ S K S V v

m m

   ] [ ] [ S K S V v

m m

 

Quasi-steady-state approach

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Two major approaches used in developing a rate expression for the enzyme-catalyzed reactions are:

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.

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EXPERIMENTALLY DETERMINING RATE PARAMETERS FOR MM TYPE KINETICS

• The values Km and Vm can be determined by

using batch reactor.

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[S0] [E0] [S] INITIAL-RATE EXPERIMENTS Product Known concentrations

The KM is the substrate concentration where vo equals one-half Vmax

Figure 14-8 Plot of the initial velocity vo of a simple Michaelis–Menten reaction versus the substrate concentration [S].

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Double reciprocal plot (lineweaver-burk plot)

• M-M equation is linearized double-reciprocal form:
• A plot of 1/v vs. 1/[S] yields a linear line with a slope of Km/Vm and y-axis

intercept of 1/Vm as in Figure 3.5 below:

• This plot gives good estimates on

Vm, but not necessarily on Km.

• 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.

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] [ ] [ S K S V v

m m

  ] [ 1 1 1 S V K V v

m m m

  

Eadie-hofstee plot

• Rearrangement of M-M equation into:
• A plot of v vs. v/[S] results in a line of slope –Km and y-axis

intercept of Vm 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].

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] [ ] [ S K S V v

m m

  ] [S v K V v

m m 



Hanes-woolf plot

• Rearrangement of M-M equation into:
• A plot of [S]/v vs. [S] results in a line of slope 1/Vm and y-

axis intercept of Km/Vm as in Figure 3.7 below:

• This plot is used to determine Vm more accurately.

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] [ ] [ S K S V v

m m

 

] [ 1 ] [ S V V K v S

m m m 



[S] [S]/v

Km/vmax

• Km

Slope=1/vmax

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].

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ENZYMES: PART 3

OUTLINE: INHIBITED ENZYME KINETICS

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INTRODUCTION

• Types of enzyme inhibitors:

– Irreversible Inhibitors (Inactivators) – Reversible inhibitors

• Competitive
• Non-competitive (mixed)
• Un-competitive

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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 (ET)

• 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

• f the enzyme or mobility restraint

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Reversible Enzyme Inhibition

• Many pharmaceuticals are enzyme inhibitors.
• Reversible inhibitors associate with enzymes

through non-covalent interactions.

• Reversible inhibitors include three kinds:

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Competitive inhibitors

• interfere with

substrate binding Uncompetitive inhibitors

• bind to ES

complex Mixed inhibitors

• bind to both E and

ES

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

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Competitive Inhibition

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Inhibition constant

    

EI I E KI 

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KM increases vmax unchanged

Competitive Inhibition

Competitive Inhibition: Lineweaver-Burk Plot

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) ] [ 1 ( ] [ ] [

max I m

K I K S S v v    Initial velocity in the presence of inhibitor

   

S K S V

M max

  

• v

 

         

I

K I 1 

MM equation:

Noncompetitive Inhibition

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The binding of the inhibitor will either alter the KM or Vmax or both. Reversible Inhibitors

Noncompetitive (mixed) Inhibition: Lineweaver-Burk Plot

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Uncompetitive Inhibition

• Uncompetitive Inhibition: Lineweaver-Burk Plot

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Km decreases vmax decreases Slope unchanged

P E ES S E  

+ I ESI KI’ k1 k2 k-1

• Competitive inhibition

 Raises KM only (intercept in L-B plot)  S and I compete for same binding site

• Noncompetitive (mixed) inhibition

 Lowers Vmax (slope in L-B plot); may increase or decrease KM  I binds at a site distinct from that at which the S binds

• Uncompetitive inhibition

 Both Vmax & KM 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

Table 14-2 Effects of Inhibitors on the Parameters of the Michaelis–Menten Equation

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EFFECT OF PH AND TEMPERATURE

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INSOLUBLE SUBSTRATE

• Enzymes are often attack large, insoluble substrate such as wood

chips (in bio-pulping for paper manufacturer) or cellulosic residues from agricultural (eg., cornstalks).

• In this case:

– Access to the reaction site on these biopolymers by enzymes is often limited by enzyme diffusion. – The no. of potential reactive sites exceeds the no. of enzyme molecules. – It is opposite the typical situation with soluble substrates where access to the enzyme’s active sites limits reaction. – Assume a slow binding of enzyme ([E] = [E0]).

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ENZYME ACTIVITY

• Only enzyme that remains catalytically active

will be measured.

• The enzyme may be denatured if it unfolds or

has its 3-dimensional shape altered by extreme pH or temperature during purification process. The denatured enzyme will have no activity.

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ENZYME UNIT

• An enzyme unit is defined as the enzyme

activity that catalyses the conversion of 1 µmol substrate into product in one minute.

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SPECIFIC ACTIVITY

• Is the number of units per milligram of protein

in the enzyme sample you added to the assay

• mixture. This is of interest as it is generally an

indication of the purity of the enzyme: the higher the specific activity, the purer the enzyme.

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IMMOBILIZED ENZYME SYSTEMS

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INTRODUCTION

• What is immobilization?
• Why Immobilize Enzymes?

– Easy separation of the enzyme from the product. – Reuse of the enzyme  cost advantage.

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METHODS OF IMMOBILIZATION

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Soluble Enzyme Immobilization Methods To Enzyme To Support Ionic Matrix Entrapment Membrane Entrapment Micro Encapsulated Between Macroscopic Membranes

LARGE SCALE PRODUCTION OF ENZYMES

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• Produced using overproducing strains of certain organisms.
• Separation and purification processes require cell disruption, removal of cell

debris and nucleic acids, percipitation of proteins, ultrafiltration of the desired enzyme, chromatographic separations, crystallization, and drying.

• The process scheme depends on whether the enzyme is intracellular or

extracellular.

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Examples of commercial enzyme

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END OF CHAPTER 3 AND CO1

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