Dr. Siham Gritly 1

Principle of Biochemistry 6-Enzymes

Course code: HFB324Credit hours: 3 hours

Dr Siham Gritly

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Terms should be learn

• allosteric enzyme; a regulatory enzyme whose affinity for its substrate is affected by the presence or absence of other molecules

• Apoenzyme; protein portion of an enzyme (i.e., lacking a coenzyme)

• Enzyme; protein which catalyzes a biochemical reaction, an enzyme name ends in -ase (e.g., amylase and carbonic anhydrase)

• Holoenzyme; complete active enzyme (i.e.,

protein + coenzyme)

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• enzyme classificationl; assignment of an enzyme to one of six groups, depending on the type of chemical reaction which the enzyme catalyzes

• first order kinetics; rate of reaction is directly proportional to the concentration of starting materials (i.e., substrate)

• Inhibition; alteration in an enzyme’s activity, usually caused by modification of the enzyme active site, so that substrate cannot bind to the enzyme, or substrate can bind but cannot be converted to product, or product cannot be released

• Michaelis-Menten kinetics; simple mathematical description of a first-order enzyme reaction [Leonor Michaelis was British and Maud Menten was Canadian]

• Catalyst; chemical substance that facilitates (or slows) a chemical process, but is unchanged by the process

• Coenzyme; small, nonprotein group attached to an enzyme; the site on the enzyme where catalysis occurs

• Cofactor; organic molecule that acts as a coenzyme• Michaelis constant ( Km ) represent the concentration of a substrate that is found in an

occurring reaction when the reaction is at one half its maximum velocity

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Enzymes • macromolecular components composed of protein.

They are known as biological catalysts responsible for supporting almost all of the chemical reactions that maintain life processes

• Enzymes are found in all tissues and fluids of the body.

• Enzymes have a high degree of specifity for types of reaction catalized and for their substrate

• Enzymes are also stereospecific catalysts for specific stereoisomers (L & D)

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• All enzymes are proteins except ribozymes;• ribozymes are certain RNA molecules act as

catalysts

• ribozymes catalyzing the cleavage and synthesis of phosphodiester bond in RNA at specific sites in RNA

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Localization of enzymes

• 1-Enzymes of the intracellular• Lysosome; enzyme required for the degradation

of complex macromolecules• Nucleous; enzymes of DNA and RNA synthesis • Cytosol; enzyme of glycolysis, fatty acid

synthesis, urea cycle, gluconeogenesis, heam synthesis

• Mitochondria; enzymes of TCA cycle, fatty acid oxidation, oxidative phosphorylation

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• 2-extracellular enzymes• Are secreted and function out from the cell • Mainly digestive enzymes • Alfa amylase secreted by salivary glands• Pepsin and renin secreted by gastric glands• Lipase, trypsin, chymotrypsin, amylase secreted by

pancrease• Aminopeptidase, dipeptidase, lactase, sucrase,

maltase, isomaltase secreted from intestinal glands

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Proenzyme or zymogen (precursor enzyme)

• Some proteolytic enzymes found in the blood or digestive tract are present in an inactive form (precursor) known as zymogen or proenzymes

• Some examples; prothrombin, proelastase, chymotrypsinogen, trypsinogen, pepsinogen which produced and stored as inactive proenzyme or zymogen

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Composition classification of enzymes

• Simple enzymes composed completely of protein

• complex enzymes, are composed of protein plus a relatively small organic molecule.

• Complex enzymes are also known as holoenzymes.

• protein component is known as the apoenzyme,

• Apoenzymes becomes active enzymes on addition of a cofacter.

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• Cofactors can be either inorganic (e.g., metal ions or organic compounds, (e.g.,favin and heme) or organic

• Organic cofactors can be either prothetic groups, which are tightly bound to an enzyme, or coenzymes, which are released from the enzyme's active site during the reaction

• Coenzymes binds apoenzyme protein molecule to produce active holoenzyme

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Cofactors can be either inorganic (e.g., metal ions or organic compounds, (e.g.,favin and heme)Organic cofactors can be either prothetic groups, which are tightly bound to an enzyme, or coenzymes, which are released from the enzyme's active site during the reaction. when the binding between the apoenzyme and non-protein components is non-covalent, the small organic molecule is called a coenzyme

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Apoenzyme, cofactor and holoenzyme

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classification and nomenclature of Enzyme

Reference; Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB) Enzyme Nomenclature

'

• Functional classification • enzymes are grouped into six functional

classes by the International Union of Biochemists (I.U.B.).

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• Class 1.Oxidoreductases- this class belong all enzymes of catalysing oxidoreduction reactions.

• The substrate that is oxidized is regarded as hydrogen donor.

• Act on many chemical groupings to add or remove hydrogen atoms

• The common name will be dehydrogenase, reductase can be used.

• Oxidase is only used in cases where O2 is the acceptor.

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• Class 2. Transferases- transfer chemical groups from one molecule to another or to another part of the same molecule.

• Kinases are specialized transferases that regulate metabolism by transferring phosphate from ATP to other molecules

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• Class 3. Hydrolases-• Add water across a bond, hydrolyzing it• These enzymes catalyse the hydrolytic

cleavage of C-O, C-N, C-C and some other bonds,

• Although the systematic name always includes hydrolase, such as digestive enzymes

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• Class 4. Lyases- remove a group from or add a group to double bonds.

• Add water, ammonia or carbon dioxide across double bonds, or remove these elements to produce double bonds

• In the common names, expressions like decarboxylase, aldolase, dehydratase (in case of elimination of CO2, aldehyde, or water) are used

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• Class 5. Isomerases-.• These enzymes catalyse geometric or

structural changes within one molecule. According to the type of isomerism (interconvert isomeric structures by molecular rearrangements),

• they may be called, cis-trans-isomerases, isomerases, tautomerases, mutases and L to D isomerase,

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• Class 6. Ligases – • Ligases are enzymes catalysing the joining together

of two molecules coupled with the hydrolysis of a diphosphate bond in ATP or a similar triphosphate

• Catalyze reactions in which two chemical groups are joined (or ligated) with the use of energy from ATP

• pyruvate oxaloacetate• enzyme = pyruvate carboxylase

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The Catalytic Activity of Enzymes

• enzymes are characterized by two fundamental properties.

• 1- First, they increase the rate of chemical reactions without themselves being consumed or permanently altered by the reaction.

• 2-Second, they increase reaction rates without altering the chemical equilibrium between reactants and products,

• a molecule acted upon by an enzyme is called substrate [S]

• substrate [S] is converted to a product (P)

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• The functional activities depend on• 1-protein portion of enzyme• 2-non-protein prothetic group or co-enzyme• Usually prothetic group is inorganic (metal ions,

Mg, Zn, Cu, Mn, Fe• Enzyme activity system may affected by;-• Negative modifiers• Change on pH• Change in enzyme concentration

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How enzyme workenergy changes during the enzymatic reaction

• All chemical reactions have an energy barrier separation reactant (S) and product (P)

• It is known as free energy of activation EA

• Free energy of activation is the energy difference between the energy of the reactant and high energy intermediates that occur during the formation of the products

• The amount of change in the free energy of a reaction is labeled ΔG

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There are two theories as to how reactions occur:

• 1- the collision theory, • it is thought that reactions occur because

molecules collide; the faster they collide, the faster the reaction occurs.

• The energy level that must be reached for the molecules to collide is called the activation energy EA Enzymes lower the activation energies so that reactions can occur quickly

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• 2- the transition state theory, • substrate are thought to form bonds and then

break bonds until they form products. • As this forming and breaking happens, free

energy increases until it reaches a transition state (also called activated complex), which is viewed as the midpoint between reactants and products.

• Reactions proceed faster if there is a higher concentration of activated complex.

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The energies of the stages of a chemical reaction. Substrates need a lot of potential energy to reach a transition state, which then convert into products.

Enzymes act by reducing the activation energy, thus increasing the rate of reaction

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If the free energy of activation EA is high, the transition state is low, and the reaction is slow.If the activation energy is lower, the reaction occurs faster because more activated complexes (transition state) can form.

this figure shows the changes in energy during conversion of a molecule of reactant or substrate S to product P through the transition state

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Transition state; in which high energy intermediates are formed during the conversion of substrate to product

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Active site of an enzyme• The catalytic activity of enzyme involves the

binding of their substrates to form an enzyme-substrate complex (ES). In an enzymatic reaction the substrate binds to a specific region of the enzyme, called the active site.

• While bound to the active site, the substrate is converted into the product of the reaction, which is then released from the enzyme.

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The active site of an enzyme lower EA and speed the chemical reaction barrier by;-

• 1-orienting substrates correctly• 2-strain substrate bonds• 3-providing a favorable micro-

environment• 4-covalently bonding to the substrate

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Enzyme substrate complex (ES complex)Enzymatic catalysis of a reaction between two substrates.

The enzyme provides a template upon which the two substrates are brought together in the proper position and orientation to react with each other

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Mechanism of enzyme action

• Formation of an enzyme-substrate complexes is the first step in enzymatic catalysis

• Substrate is bound through multiple non-covalent interactions at the active site of the enzyme forming substrate complex which is then converted to product and free away enzyme

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• Two model of substrate binding to the active site of the enzyme;

• -lock and key model

• - induced fit model

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lock and key modelthe substrate and enzyme active site have complementary shapes

in which the substrate fits exactly into the active site

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induced fit model; the configurations of both the enzyme and substrate are modified by substrate binding

This model proposes that the initial interaction between enzyme and substrate is relatively weak, but that these weak interactions

rapidly induce conformational changes in the enzyme that strengthen binding

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Enzyme Kinetic

• Enzyme kinetics are the study of reaction rates and how they change in response to changes in experimental parameters.

• The rate of reaction affected by;• -the amount of substrate present (S)• -the effect on initial velocity (Vₒ) of varying

substrate concentration when enzyme concentration is held constant

• initial velocity is the rate of reaction as soon as enzyme and substrates are mixed

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Effects of substrate concentration on the initial velocity of an enzyme catalyzed reaction keeping enzyme concentration

constant

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• Low concentrations of substrate, initial velocity (Vₒ) increases linearly with an increase in S this condition known as first order kinetics

• At higher substrate concentration, (Vₒ) increases by smaller amount in response to increase in S

• That is, The velocity of an enzyme-catalyzed reaction increases as the concentration of the substrate increases

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• When there are small increase in (Vₒ) with increasing S a condition known as zero order kinetics and a plateau is

called maximum velocity vmax (when all active sites on the enzyme are filled with substrate)

• That is to say; At saturation levels of substrate, the enzyme functions at its maximum velocity (vmax)

• the occurrence of higher concentration of substrate cannot increase the velocity further

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Factors affecting the Velocity of Enzyme reaction

• Any substrate that affects the configuration of an enezyme affects its activity

• Various factors that affect enzyme activity are;-• 1-substrate concentration• 2-Enzyme concentration• 3- Ph (H ions concentration)• 4-temperature• 5-product concentration• 6-inhibitors

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Michaelis-Menten constant Km

• Michaelis constant ( Km ) represent the concentration of a substrate that is found in an occurring reaction when the reaction is at one half its maximum velocity (½ Vmax)

• Km is an inverse measure of the strength of binding between the enzyme and its substrate. The lower the Km, the greater the affinity

• E+S ↔ ES-------1• ES→ E+ P ---------2

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If an enzyme has a high Km value then the abundance of substrate must be present to raise the rate of reaction to half its maximum the enzyme has a low affinity for its substrate. e.g, glucokinase the low affinity of glucokinase for glucose prevents too much glucose being removed from the blood during period of fasting

substrate concentration versus reaction velocity

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The Michaelis-Menten Equation represent the concentration of a substrate that is found in an occurring reaction when the

reaction is at one half its maximum velocity (½ Vmax)

Vₒ= initial reaction velocity is the rate of reaction as soon as enzyme and substrates are mixedVmax = maximum velocity is observed when all active sites on the enzyme are filled with substrate Km = Michaelis-Menten constant is the substrate concentration at which the reaction rate is half of its maximum velocity Vmax[S ] = substrate concentration

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Michaelis constant has two significance

• 1-Km is the concentration of substrate at which half the active site of enzymes are filled, thus Km provide measure of the substrate concentration required for the reaction to occur

• 2-Km is the measure for the strength of the ES complex or the affinity of enzyme to substrate

• -a high Km indicates weak binding with its substrate

• -a low Km indicates strong affinity or binding to substrate

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The Effects of Enzyme InhibitorsReference; Michael W King, PhD | © 1996–2012 themedicalbiochemistrypage.org, LLC | info @ themedicalbiochemistrypage.org

• Competitive Inhibitor;- Inhibitor at the catalytic site, where it competes with substrate for binding in a dynamic equilibrium- like process. Inhibition is reversible by substrate

• Kinetic effect; Vmax is unchanged; Km, as defined by [S] required for ½ maximal activity, is increased

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• Noncompetitive Inhibitor;- Binds E or ES complex other than at the catalytic site. Substrate binding unaltered, but ES complex cannot form products. Inhibition cannot be reversed by substrate.

• Kinetic effect; Km appears unaltered; Vmax is decreased proportionately to inhibitor concentration

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Reference; Michael W King, PhD | © 1996–2012 themedicalbiochemistrypage.org, LLC | info @ themedicalbiochemistrypage.org

• The characteristic of all the reversible inhibitors is that when the inhibitor concentration drops, enzyme activity is regenerated.

• Usually these inhibitors bind to enzymes by non-covalent forces and the inhibitor maintains a reversible equilibrium with the enzyme.

• The equilibrium constant for the dissociation of enzyme inhibitor complexes is known as Ki

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Competitive Inhibitor

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noncompetitive Inhibitor

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Regulatory enzymes

• In each enzyme system, however, there is at least one enzyme that sets the rate of the overall sequence because it catalyzes the slowest or rate-limiting reaction.

• These regnlatory enzymes exhibit increased or decreased catalytic activity in response to certain signals.

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Allosteric Enzymes

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Allosteric Enzymes Are Regulated by Noncovalent Binding of Modulators

• allosteric regulation is the regulation of an enzyme or other protein by binding an effector molecule at the protein's allosteric site (that is, a site other than the protein's active site)

• Effectors that enhance the protein's activity are referred to as allosteric activators,

• whereas those that decrease the protein's activity are called allosteric inhibitors

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• Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB) Enzyme Nomenclature

• Michael W King, PhD | © 1996–2012 themedicalbiochemistrypage.org, LLC | info @ themedicalbiochemistrypage.org

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