Enzyme (2)

• Every living cell performs continuously thousands of different chemical reactions.

• Taken nutrients are transformed into a multitude of cell specific components. In this way, sugars, amino acids and their precursors, organic acids, nucleotides, lipids and other substances are produced

• The totality of these reactions is summarized as the cell metabolism

• The sum of all biochemical processes, consists of both catabolic and anabolic processes.

CatabolismCatabolism

• comprises all processes, in which complex molecules are broken down to simpler ones

• energy is released during catabolic reactions.

• involve oxidation and hydrolysis.

• Example-cellular respiration• - hydrolysis lipid

Anabolic reactionsAnabolic reactions

• Anabolism involves the synthesis of complex molecules from simpler molecules

• requires energy input.• Involved condensation process• Example:Synthesis of a protein from

amino acids and synthesis of a polysaccharide from monosaccharides are examples of anabolic pathway.

• Exergonic reactions are reactions that liberate more energy than they take in.

• During an anabolic reaction, energy input is required to synthesise complex molecules from simpler molecules.

• The products contain more potential energy than the reactants.

• Endergonic reactions take in more energy than they liberate

• During catabolic reactions, the complex molecules break down into simplemolecules with the release of energy

• The products have less potential energy than the reactants.

• The activation energy, EA is the amount of energy required to bring the reactants into the correct position to interact (transition state).

Metabolic PathwayMetabolic Pathway

• All metabolic reaction are catalysed by enzymes

• A metabolic pathway is a number of reactions catalysed by a sequence of enzymes

• The reaction occur in a sequence

• Each step catalysed by specific enzymes

• Intermediate can be used as starting point

• A biochemical reaction pathway consist of a series of enzyme catalysed reaction

• The product of each reactions becomes the substrate of the next reaction

• Some of metabolic pathway are cyclic

EnzymesEnzymes• Enzymes are biological catalysts produced by living

cells. • Enzyme lower the amount of activation energy

needed. • They speed up the rate of biochemical reactions in

the cell but remain unchanged at the end of the reactions.

• Most enzymes are globular protein molecules. • The chemical which an enzyme acts on is called its

substrate. • The function of the enzyme depends on their three-

dimensional structure.• very small amount qf enzymes is needed to react

with a large amount substrate.

• Enzymes are highly specific in action.• Enzymes possess active sites and nil only

catalyse a reaction when the substrate and active site have complementary shapes.

• For example, catalase catalyses the breakdown of hydrogen peroxide to water and oxygen.

• The reactions catalysed by enzymes are usually reversible,

• The enzyme combines with its substrate to form an enzyme-substrat complex.

• The complex then breaks up into product and enzyme.

• Enzymes themselves remain unchanged at the end of the reaction. Hence, only a small amount of an enzyme is needed and it can be ed repeatedly.

• enzyme is specific for a particular reaction because its amino sequence is unique and causes it to have a unique three- dimensional structure called the active site.

Chemical reactionsChemical reactions

• Chemical reactions need an initial input of energy = THE ACTIVATION ENERGY

• During this part of the reaction the molecules are said to be in a transition state.

© 2007 Paul Billiet ODWS

Reaction pathwayReaction pathway


Enzyme structureEnzyme structure

• Enzymes are proteins

• They have a globular shape

• A complex 3-D structure

Human pancreatic amylase

The active siteThe active site

• One part of an enzyme, the active site, is particularly important

• The shape and the chemical environment inside the active site permits a chemical reaction to proceed more easily

© H.PELLETIER, M.R.SAWAYA ProNuC Database


The The substratesubstrate

• The substrate of an enzyme are the reactants that are activated by the enzyme

• Enzymes are specific to their substrates

• The specificity is determined by the active site


• When a substrate molecule collides into an enzyme, it fits into a depression on the surface of the enzyme molecule.

• This depression is called the active site. • A reaction takes place and the product leave the

active site,• enzyme freely to receive another substrate

molecule• The active site has a specific shape to which

only one kind substrate will fit which explains why enzymes are specific in then action.

• A substrate has a surface region that is complementary in size shape, solubility and charge to the active site.

• The minimum energy required for substances to react is called activation energy (Ea) or free energy of activation

• This energy is required to break or make bonds.

• It can be provided in the form of heat that the substrates absorb from the surroundings-kill cell

• Use enzyme-speed up the reaction rate-lower the activation energy-reaction can take place

Making reactions go fasterMaking reactions go faster

• Increasing the temperature make molecules move faster

• Biological systems are very sensitive to temperature changes.

• Enzymes can increase the rate of reactions without increasing the temperature.

• They do this by lowering the activation energy. • They create a new reaction pathway “a short

cut”


An enzyme controlled An enzyme controlled pathwaypathway

• Enzyme controlled reactions proceed 108 to 1011 times faster than corresponding non-enzymic reactions.


• Enzymes speed up the reaction rate by lowering the activation energy.

• The lower the activation energy, the easier it is for the reaction to take place.

• High activation energy means that the substrate molecules must collide with the enzyme molecules very strongly in order to react.

• So, reactions with high activation energy proceed slowly at low temperatures where most molecules move relatively slow.

• Without enzymes, most of the biochemical reactions in living cells at body temperature would occur very slowly or not at all.

• Enzymes are highly specific in

• (a) the reaction catalysed

• (b) their choice of reactants

Mechanism of action and KeneticMechanism of action and Kenetic

• Lock and Key hypothesis

• Induced fit hypothesis

Lock and Key hypothesisLock and Key hypothesis

• Proposed by Emil Fisher

• Enzyme has 3 D shape

• An enzyme is a large globular protein with a specific three-dimensiona shape.

• It has a groove called the active site containing amino acid side chains

• active site-complementary to that substrate

• the shape of the substrate (‘key’) fits into the rigid active site of the enzyme (‘lock’) forming an enzyme-substrate complex

• Reaction takes place and products are formed released.

The Lock and Key HypothesisThe Lock and Key Hypothesis

• Fit between the substrate and the active site of the enzyme is exact

• Like a key fits into a lock very precisely• The key is analogous to the enzyme and the substrate

analogous to the lock. • Temporary structure called the enzyme-substrate

complex formed • Products have a different shape from the substrate • Once formed, they are released from the active site • Leaving it free to become attached to another substrate



Enzyme may be used again

Enzyme-substrate complex

E

S

P

E

E

P

Reaction coordinate© 2007 Paul Billiet ODWS


• This explains enzyme specificity

• This explains the loss of activity when enzymes denature


The induced-fit hypothesisThe induced-fit hypothesis

• the active site is flexible

• NOT exactly complementary to the shape of the substrate.

• An enzyme collides with the substrate molecule. The substrate bind the active site.

• induces a slight change in the shape of the enclose the substrate- make fit more precise

• The active site becomes fully complementary with the substrate as the substrate bind the enzyme

• The active site is not an exact fit for the substrate.

• The enzyme and its active site are flexible. • When the substrate enters the active site it

induces a small change in the shape of the enzyme.

• The amino acids which make up the active site are moulded into a precise shape complementary to the substrate.

• This enables the enzymes to carry out their catalytic function.

Michaelis-Menten KineticMichaelis-Menten Kinetic

• A reaction model was proposed by Michaelis and Menten to account for enzyme-catalysed reactions.

• In the model the enzyme binds reversibly with its substrate to form an enzyme-substrate complex that breaks down to product.

• In second step- enzymecatalyses the chemical reaction and form product

• The enzyme is regenerated.

• The close fit brings the molecules in close proximity and in the correct orientation for reaction to take place.

• causes stressing and distortion of chemical bonds of the substrates

• causes the bonds to break and new bonds to form. • makes it easier for the substrate to be changed into the

product thus, lowering the activation energy required.• the products formed have a different shape and are

released from the enzyme. • the enzyme structure is unchanged and can be reused.

The Induced Fit HypothesisThe Induced Fit Hypothesis

• This explains the enzymes that can react with a range of substrates of similar types

Hexokinase (a) without (b) with glucose substratehttp://www.biochem.arizona.edu/classes/bioc462/462a/NOTES/ENZYMES/enzyme_mechanism.html


The Induced Fit HypothesisThe Induced Fit Hypothesis

• Some proteins can change their shape (conformation)

• When a substrate combines with an enzyme, it induces a change in the enzyme’s conformation

• The active site is then moulded into a precise conformation

• Making the chemical environment suitable for the reaction

• The bonds of the substrate are stretched to make the reaction easier (lowers activation energy)


Enzyme kineticsEnzyme kinetics

• is the study of the rate at which an enzyme works.

• Theory enzyme kinetics- Michaelis Menten –Kenetic

• Rate of enzyme reaction-measuring the rate of formation of product (s)

• the rate is influenced by several factors:

• (a) The concentration of substrate molecules

• (b) Temperature

• (c) Presence of competitive and non-competitive inhibitors

• (d)pH

Michaelis-Menten equationMichaelis-Menten equation

• A reaction model was proposed by Mechaelis and Menten

• To account for enzyme-catalysed reaction

• The enzyme bind reversibly with it substrate to form an enzyme-substrate complex

• Enzyme substrate breaks down to product

• The enzyme is regenerated

• The velocity of the reaction is determined by Michaelis-Menten formula i.e.

where VM is equal to maximum velocity and [S] is the substrate concentrationThe Michaelis – Menten equation show how reaction velocity varies with the substrate concentration

• The usual approach is how the velocity of the reaction varies with changes in concentration of the substrate

• When the velocity of the reaction is plotted against the substrate concentration-the curve is obtained

Enzyme kineticsEnzyme kinetics

• From the graph-

• The velocity of reaction is proportional to concentration initially

• When the substrate is added-the velocity become maximum

• The enzyme molecule are saturated with the substrate molecule

• From the graph, Michaelis-Manten constant (Km) can be determined.

• Km is the substrate concentration when the velocity of the reaction is half maximum.

• The constant is fixed for a particular enzyme under a certain condition.

• Km is inversely proportional to the affinity of an enzyme to its substrate.

• A very small Km for an enzyme indicates its high affinity for substrate and it is a measure of its efficiency i.e. it can work very fast.

• The Michaelis-Menten equation shows how reaction velocity varies substrate concentration.

Steady State AssumptionSteady State Assumption

• The M-M equation was derived in part by making several assumptions.

• An important one was: the concentration of substrate must be much greater than the enzyme concentration.

• Therefore, it follows that the rate of ES formation will be equal to the rate ES breakdown.

• At V max-all the active site saturated with substrate-number of ES= Total amount of enzyme

• The total concentration of substrate is high enough

• The back reaction E + P is negligible• The rate of formation of ES is assumed to be

the same as the rate of breakdown of E + S• Total active site= empty site + occupied sites

• At rate k1

• -enzymes will combine with the substrate

To form enzyme substrate complex

• enzyme substrate complex form E + S at rate k-1

• enzyme substrate complex to form product at rate k2

• Substrate Saturation of an Enzyme

Lineweaver- Burk plotLineweaver- Burk plot

• Is useful to determine Km and Vm.

• A plot is obtained-plotting the reciprocal of V versus the reciprocal of substrate concentration

• An easier and more accurate method is by using relatively fewer data points to plot the reciprocals of the velocity and substrate concentration against one another.

• When this is done, a straight line is obtained.

• This is known as the Lineweaver-Burk plot.

• We can determine KM and VM from the reciprocal of Michaelis-Menten formula as follow:

• To get the value of KM.

• assume

• To get Vm, assume

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Enzyme (2)