At the end of this topic, students should be able to: Explain the properties of an enzyme. Describe the enzymatic reaction. Describe factor that influence the activity of enzymes. Explain basic principles of enzyme inhibition. Describe allosteric regulation of enzyme. Define the classes of enzymes.
1. Enzymes are proteins that act as biological catalysts Catalyze nearly all of the chemical reactions that take place in the body Reaction with enzymes will lower the activation energy
2. Have unique (globular protein)3-D conformations. The specificity of an enzyme results from its shape, which is a consequence of its amino acid sequence. 3. Have active site for substrate binding.  Active site - The location where a substrate binds with intermolecular forces to an enzyme. Active site contains amino acid side chain that is complementary to the substrate. Substrate - the reactant of a chemical reaction.
4. Reaction reversible - catalyzing reaction of enzyme is reversible. Enzymes increase the rate of a reaction, but are unchanged themselves at the end of the reaction 5. Enzyme specificity For any chemical reaction, the reactant that an enzyme acts on is called the enzyme’s substrate The enzyme binds to its substrate, forming an enzymesubstrate complex
5. Very efficient - small amount of enzyme can change a large amount of substrate into products. 6. Affected by many factors such as by pH, temperature, substrate and enzyme concentration.
A restricted region of the enzyme molecule which binds to the substrate. This region is typically a pocket or groove on the surface of the protein. Precisely fits the contours of a substrate via weak electrostatic interactions & facilitates bond reactivity. Enzyme-Substrate Binding
Also called a regulatory site. A position other than the active site of an enzyme. Allosteric activator and allosteric inhibitor can attach here. Substrate cannot attach here.
ď ą Notice that without the enzyme, it takes a lot more energy for the reaction to occur. By lowering the activation energy, you speed up the reaction.
The reactants AB and CD must absorb enough energy from the surroundings to reach the unstable transition state, where bonds can break.
Bonds break and new bonds form, releasing energy to the surroundings.
Activation energy is the minimum energy necessary for a specific chemical to occur OR the amount of energy required for reactants to reach the transition state before changing into the product.
Enzymes Reduce Activation Energy 
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Enzymes lower the amount of activation energy needed to initiate a reaction and speed up the reaction. Enzymes can bind to the substrate (reactant) to form ES complex. The ES state has lower energy than the transition state in uncatalysed(without enzyme) reaction.
The chemical which an enzyme works on is called substrate. An enzyme combines with substrate to form a short-lived enzyme/substrate complex. Once a reaction has occurred, the complex breaks up into products and the enzyme is free to receive another. substrate.
E+S
ES
EP
E+P
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P
+
S
+
S
P
E
+
S
ES complex
E
+
P
(a) Exergonic reaction: energy released
△G = [E of product ] – [E of reactant] Reactants
2 types of enzymatic reaction :-
- chemical reaction in which energy is released. -
- breakdown of high energy substances to low energy product -
Amount of energy released (G 0)
Free energy
a) Exergonic reaction
Energy Products
Progress of the reaction
Endergonic reaction
(b) Endergonic reaction: energy required,
- chemical reaction in which energy is formed.
- simpler substances to complex substances
Products
Energy coupling – Energy generated by catabolic process used by cells to perform anabolic process.
Free energy
Energy Reactants
Progress of the reaction
Amount of energy required (G 0)
How enzyme lowers the activation energy •
•
In an enzymatic reaction, the substrate binds to the active site of the enzyme. The active site can lower an EA barrier by(factors): –
–
–
Orienting substrates correctly for substrate to come together in proper orientation for a reaction to occur between them. Straining substrate bonds stretch substrate toward their transition state conformation, stressing and bending critical chemical bonds that must be broken Providing a favorable microenvironment If the active site has amino acid with acidic R group active site maybe a pocket of low pH compared to the neutral cell.
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(1)Early theory: lock-and-key model.
Enzyme had a particular shape in which substrate fits exactly. Substrate is imagined as a key whose shape is complementary to the enzyme as a lock. correctly sized key (substrate) fits into the key hole (active site) of the lock (enzyme) which has a specific shape. Substrate reacts to form product. Product is released.
Lock and Key Model
SPECIFIC SHAPE
(2)Current theory: induced fit model. Enzyme structure is flexible, not rigid Enzyme and active site adjust shape to bind with substrate. A slight change in the shape of an enzyme’s active site is induced by the substrate. Increases range of substrate specificity. Shape changes also improve catalysis during reaction. Theory widely accepted today.
Induced Fit Model
The substrate enters the active site. They bind and the enzyme changes shape, forming the enzyme-product complex. Pressure on the substrate bonds lowers the activation energy causing an increased reaction rate.
TEMPERATURE
ENZYME ACTIVITY AFFECTORS
pH
SUBSTRATE CONCENTRATION
ENZYME CONCENTRATION
Temperature
Little activity at low temperature Rate increases with temperature Most active at optimum temperature (usually 37°C in humans) Activity lost with denaturation at high temperature
Denaturation – A change in the shape of an enzyme, which, in turn, affects the shape of the active site. This compromises the ability of the enzyme to act on a substrate. This is often caused by changes in temperature or pH.
pH 27
Maximum activity at optimum pH R groups of amino acids have proper charge Tertiary structure of enzyme is correct Narrow range of activity Most lose activity in low or high pH
Substrate Concentration 

Increasing substrate concentration increases the rate of reaction (enzyme concentration is constant) Maximum activity is reached when all of the enzymes combine with substrate
Enzyme Concentration
An enzyme must bind with a substrate and form an enzyme substrate complex before products can be produced.
The rate of an enzyme-catalysed reaction is directly proportional to the [E] if the substrates are present in excess concentration and no other factors limiting.
The graph represents unlimited substrate and unlimited enzyme.
ENZYME INHIBITION
COMPETITIVE INHIBITOR
REVERSIBLE
NON COMPETITIVE INHIBITOR
REVERSIBLE
IRREVERSIBLE
Cause a loss of catalytic activity Change the protein structure of an enzyme May be competitive or noncompetitive Some effects are irreversible
Competitive Inhibition
Has a structure similar to substrate. I LIKE ACTIVE SITE TOO!!! Occupies active site. Competes with substrate for active site. The inhibitor is assumed to bind to the free enzyme but not to the enzyme-substrate (ES) complex. Has reversed effect by increasing substrate concentration. E.g. Malonate is a competitive inhibitor of the enzyme succinate dehydrogenase.
Competitive Inhibition
OHHH,I LOVE AS TOO!!
Noncompetitive Reversible Inhibition
This type of inhibitor has no structural similarity to the substrate and combines with the enzymes at other site than its active site. I DON’T LIKE AS,UHHH!!!
Substrate binding unaltered, but Enzyme-Substrate-Inhibitor complex cannot form products. It does not affect the ability of the substrate to bind with the enzyme, but makes it impossible for catalysis to take place. But, once the inhibitor release from the enzyme, the enzyme reaction can be active again (reversible).
Noncompetitive Irreversible Inhibition Combines with the enzymes at other site than its active site, altered the enzymes structure. Leaves enzymes permanently damaged. Enzyme unable to carry catalytic reaction. Breaks disulphide bonds that maintain enzyme shape; once these bonds break the enzyme structure changes forever – loss of function. E.g. heavy metal ions like mercury (Hg 2+), silver (Ag+) and arsenic (As+), or certain iodinecontaining compounds completely inhibit some enzymes.
Substrate A substrate can bind normally to the active site of an enzyme.
Active site Enzyme
(a) Normal binding A competitive inhibitor mimics the substrate, competing for the active site.
Competitive inhibitor
(b) Competitive inhibition
A noncompetitive inhibitor binds to the enzyme away from the active site, altering the conformation of the enzyme so that its active site no longer functions.
Noncompetitive inhibitor
(c) Noncompetitive inhibition
Allosteric regulation - Occurs when a nonsubstrate molecule binds or modifies a site other than the active site of an enzyme (called the allosteric site), thereby inducing the enzyme to change its shape.  The shape change can result in the activation or inactivation of an enzyme. 
Most allosterically regulated enzymes are constructed from 2 or more polypeptide chains, or subunits. Each subunit has its own active site. The entire complex oscillates between 2 conformational states – catalytically active and inactive. An activating or inhibiting regulatory molecule binds to a regulatory site (allosteric site), often located where subunits join.
The binding of an activator to a regulatory site stabilizes the conformation that has functional active sites. The binding of an inhibitor stabilizes the inactive form of the enzyme. The subunits of an allosteric enzyme fit together and the conformational change in one subunit is transmitted to all others. This interaction of subunits, a single activator or inhibitor molecule that binds to one regulatory site will affect the active sites of all subunits.
Allosteric activator stabilizes active form
Allosteric inhibitor stabilizes inactive form
(a) Allosteric activators and inhibitors. In the cell, activators and inhibitors dissociate at low concentrations. The enzyme can then oscillate again.
Binding of one substrate molecule to active site of one subunit locks all subunits in active conformation.
(b) Cooperativity: another type of allosteric activation. Note that the inactive form shown on the left oscillates back and forth with the active form when the active form is not stabilized by substrate.
End-product inhibition (negative feedback inhibition) Feedback inhibition - A method of metabolic control in which the end product of a metabolic pathway acts as an inhibitor of an enzyme within that pathway. When the end product of a metabolic pathway begins to accumulate, it may act as an allosteric inhibitor of the enzyme controlling the first step of the pathway. Thus, the product starts to switch off its own production as it builds up. The process is selfregulatory.
As the product is used up, its production is switched back on again. This is called end-product inhibition and is an example of a negative feedback mechanism. In this way, feedback inhibition thereby prevents the cell from wasting chemical resources by synthesizing more molecules than necessary.
Figure: Feedback inhibition in isoleucine synthesis
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Nonprotein substances that bound tightly or loosely and are essential for some enzymes to function efficiently. Enzyme cofactors
Inorganic ions
Inorganic ions that attach temporarily Inorganic ions such as Zn2+ Ca2+, Mg2+, K+, Fe2+ and Na+, Cl- Zn2+ (the cofactor for carbonic anhydrase), Cl- (the cofactor for salivary amylase)
Prosthetic groups
Non-protein organic substance that binds tightly and permanently -Cytochrome oxidase has prosthetic group heme. -Enzyme Catalase (heme) catalyses hydrogen peroxide into O2 + H20
Coenzymes -Bind loosely and temporarily to an enzyme (organic). -Will be released from the enzyme’s active site during reaction. -Transfer agents (transfer electrons, atoms from 1 enzyme to another. -Many coenzymes are obtained from vitamin - Ex: NAD+ (vitamin B) Ex: FAD
Enzyme –protein portion (apoenzyme) + Nonprotein component (cofactors)
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haloenzyme
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End in –ase Identifies a reacting substance sucrase – reacts sucrose lipase - reacts lipid Describes function of enzyme oxidase – catalyzes oxidation hydrolase – catalyzes hydrolysis Common names of digestion enzymes still use –in pepsin, trypsin pepsin
TYPE
REACTION CATALYZED
TYPICAL REACTION
Oxidoreductase
are involved in oxidation/reduction.
AH + B BH (reduced)
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ENZYME A+
- Transfer of H and O atoms or AO electrons from one substance to A + O (oxidized) another
Dehydrogenase -Removal of H atom from substrate Oxidase -Addition of O to H with the formation of water Ex: Cyctochrome oxidase, Lactate dehydrogenase
Transferase
Transfer of a functional group from one substance to another - Group may be methyl-, acetyl, -amino- or phosphate group.
AB + C BC
A+
Transaminase kinase Alanine deaminase Acetate kinase
Hydrolases
Formation of two products from a substrate by hydrolysis (addition of water)
AB + H2O → AOH Lipase, amylase, sucrase + BH
TYPE
REACTION CATALYZED
TYPICAL REACTION
ENZYME
Lyases
Non – hydrolytic addition or removal of groups from substrates. -catalyze certain reactions in which double bonds form and break
RCOCOOH RCOH + CO2
Decarboxylase Aldolase Isocitrate lyase
Isomerase
Intramolecules rearrangement
AB → BA
Glucosephosphate isomerase
- Rearrangement of atoms within a molecule
Ligases
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Join together two molecules by simultaneous breakdown of ATP.
X + Y+ ATP Acetyl-Co A →XY + ADP + Pi synthetase, Phospho kinase, DNA ligase
TUTORIAL 1. 2. 3. 4.
What are allosteric sites? Define enzyme. What is enzyme-product complex? What is the optimal pH for pepsin and salivary amylase? Why do they differ?
TUTORIAL 5. State two ways in which pH affects the rate of an enzymatic reaction. Discuss the effect of temperature in enzymatic reaction. 6. Why does the reaction rate become constant even when substrate concentration continues to increase?