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• 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
© 2007 Paul Billiet ODWS
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
© 2007 Paul Billiet ODWS
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
© 2007 Paul Billiet ODWS
• 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”
© 2007 Paul Billiet ODWS
An enzyme controlled An enzyme controlled pathwaypathway
• Enzyme controlled reactions proceed 108 to 1011 times faster than corresponding non-enzymic reactions.
© 2007 Paul Billiet ODWS
• 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
© 2007 Paul Billiet ODWS
The Lock and Key HypothesisThe Lock and Key Hypothesis
Enzyme may be used again
Enzyme-substrate complex
E
S
P
E
E
P
Reaction coordinate© 2007 Paul Billiet ODWS
The Lock and Key HypothesisThe Lock and Key Hypothesis
• This explains enzyme specificity
• This explains the loss of activity when enzymes denature
© 2007 Paul Billiet ODWS
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
© 2007 Paul Billiet ODWS
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)
© 2007 Paul Billiet ODWS
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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