The Nature Of Enzymatic Catalysis

LECTURE 5

The nature of enzymatic catalysis Biology, Campbell & Reece: Ch8, 148-157

By

Dr Mohamed Abumaree Molecular Reproductive Biology & Immunology College of Medicine King Saud bin Abdulaziz University for Health Science Riyadh 2009 1

Chemical Reactions

Exergonic Reaction

Endergonic Reaction

Exergonic Reaction

Endergonic Reaction

Spontaneous Chemical Reaction

Non–Spontaneous Chemical Reaction

Release of free energy

Free energy absorbed & stored

G decreases

G increases

ΔG is negative

ΔG is positive

Magnitude of ΔG: maximum amount of work a reaction can perform

Magnitude of ΔG: quantity of energy required to drive a reaction

The greater the decrease in free energy, the greater the amount of work that can be done

Cell Works 1.Mechanical Work (such as, muscle cell contraction) 2.Transport Work (such as substance across membranes against the direction of spontaneous movement) 3.Chemical Work (such as polymer synthesis) 4

Cell Work  Cells use ATP to mediate energy coupling (the use of exergonic process to drive an endergonic process)  ATP hydrolysis releases energy greater than the energy released by other molecules 5

ATP Structure

ATP contains ribose, adenine & three phosphate groups

ATP Hydrolysis The break of bonds between phosphate groups yields Pi & ADP

The reaction is exergonic releases 7.3 kcal of energy/ mole of ATP hydrolyzed

Why ATP hydrolysis releases so much energy? 1.Three negatively charged phosphate groups in ATP molecule 2. So, charges are crowded together!! 3. Their repulsion instable ATP molecule

ATP Performs Work

 ATP hydrolysis generates insufficient energy to perform cellular works  So, enzymes couple energy generated from ATP hydrolysis to endergonic reactions by phosphorylating a reactant, which is more reactive (less stable) than the unphosphorylated reactant 9

ATP Regeneration

ATP cycle couples the exergonic processes to the endergonic processes

The Activation Energy Barrier  Energy required to twist & change the bonds of reactant molecules in order to push them over an energy barrier (hill) to begin the downhill part of the reaction

 EA provides a barrier to determine the rate of the reaction  Reactants must absorb enough energy to reach the top of EA barrier before the reaction occurs  Some reactions, EA is modest at room temperature so there is sufficient energy for the reactants to reach the transition state in a short time…But, in most cases, EA is high so the transition state cannot be reached so the reaction doesn’t occur… So, the reaction will occur only if the reactants are heated 13

How Does Enzymes Lower EA Barrier?  Macromolecules (rich in free energy), decompose spontaneously at temperatures typical for cells, but, the activation barriers must be overcome for cells to carry out the metabolic reactions necessary for life  Heat speeds a reaction by allowing reactants to achieve the transition state, but this is inappropriate for the biological systems, for the following reasons: 14

1. High temperature denatures proteins so kills cells 2. Heat will speed up all reactions, not only the necessary ones!!  Therefore, cells use enzymes to catalyze a reaction  Enzymes lower EA barrier to enable the reactant molecules to absorb enough energy  Thus reaching the transition state at moderate temperatures  Enzymes speed up reactions that occur eventually anyway 15

1. Enzymes determine (Selective) which chemical processes will go on in the cell at a specific time 2. Enzyme can not change ΔG for a reaction; it can not make an endergonic reaction exergonic

Enzyme Specificity  Enzyme binds specifically to its substrate (substrates) forming an enzyme substrate complex  The enzyme catalytic action converts the substrate to the product ( products) of the reaction

Molecular Recognition Enzymes (proteins) has an active site (a pocket) on the surface of the protein, where the substrate binds Active site is formed by a few amino acids, acids while the rest of the protein molecule provides a framework determining the active site configuration

The specificity of an enzyme due to the compatible fit between the shape of its active site and the shape of the substrate The active site is not a rigid receptacle (container) for the substrate

Catalysis in Enzyme Active Site

The entire cycle happens so fast

 Very small amounts of an enzyme can have a huge metabolic impact by functioning over & over again in catalytic cycles  Enzyme can catalyze the forward & the reverse reactions 

Which reaction succeeds depends concentrations of reactants & products

on

the

 Enzyme always catalyzes a reaction in the direction of equilibrium 21

 The active site provides a template for the substrates to come together in the proper orientation so a reaction can occur between them  The active site distorts the substrate to approach the transition state  Thus reduces the amount of free energy that must be absorbed to achieve a transition state  The active site may also microenvironment, microenvironment such as pH

provide

a

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 The rate at which a specific amount of enzyme converts substrate to product is partly a function of the initial concentration of the substrate: substrate • The more substrate molecules are available, available the more frequently they access the active sites of the enzyme molecules  However, there is a limit to how fast the reaction can be pushed by adding more substrate to a fixed concentration of enzyme 23



When the concentration of the substrate is high enough, all enzymes’ active sites will be engaged (Enzyme is saturated ) (E  When the product exits an active site, another substrate molecule enters  The reaction rate is determined by the speed at which the active site converts substrate to product  When an enzyme is saturated, saturated the rate of product formation is increased by adding more enzymes 24

Effects of Local Conditions on Enzyme Activity  The activity of an enzyme (how efficiently the enzyme functions) is affected by: 1) Temperature 2) pH 3) Chemicals that specifically influence enzyme function 25

Effects of Temperature The rate of an enzymatic reaction increases with increasing temperature  Because substrates crash with active sites more frequently when the molecules move rapidly  Each enzyme has an optimal temperature at which its reaction rate is greatest 26

Above the optimal temperature of the enzyme, the speed of the enzymatic reaction drops sharply  The thermal disturbance of the enzyme molecule disrupts the bonds (hydrogen & ionic bonds & other weak interactions) that stabilize the active conformation, then the protein molecule eventually denatures 27

Most human enzymes have optimal temperatures of about 35–40°C (close to human body temperature)  Without denaturing the enzyme, the optimal temperature allows the greatest number of molecular collisions & the fastest conversion of the reactants to product molecules 28

Effects of pH  Enzyme has an optimal pH (pH 6– 8) in which enzyme is most active  Exceptions:  Pepsin (a digestive enzyme in the stomach) works best at pH 2  Trypsin (a digestive enzyme in the intestine) works best at pH 8 29

Cofactors  A nonprotein molecule or ion (zinc, iron & copper) that is required for the enzyme function  Can be permanently bound to the active site or may bind loosely & reversibly with the substrate during catalysis  If the cofactor is an organic molecule, it is called a coenzyme, coenzyme such as vitamins 30

Enzyme Inhibitors  Some chemicals selectively inhibit enzyme function  If the inhibitors bind to the enzyme by covalent bonds, bonds inhibition is usually irreversible, irreversible but many inhibitors bind weakly to the enzyme so the inhibition is reversible

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 Some reversible inhibitors resemble the normal substrate molecule & compete for admission into the active site  This is called competitive inhibitors  The competitive inhibition can be overcome by increasing the concentration of substrate  So the active sites become available, more substrate molecules than inhibitor molecules are around to gain entry to the sites 32

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  Toxins & poisons are irreversible enzyme inhibitors. For example, sarin (a nerve gas) causes death  Not all inhibitors are harmful, natural inhibitors (selective inhibition) regulate enzyme activity to control cellular metabolism 33

Allosteric Regulation of Enzymes Regulatory Molecules 

Naturally regulate enzyme activity (Reversible; noncompetitive inhibitors)  They bind to a site elsewhere on the enzyme, enzyme via noncovalent bonds; thus change the shape and the function of its active site 34

Allosteric regulation The function of a protein at one site is affected by the binding of a regulatory molecule to a separate site resulting in inhibition or stimulation of the activity of an enzyme

Feedback Inhibition In feedback inhibition, a metabolic pathway is switched off by the inhibitory binding of its end product to an enzyme that acts early in the pathway 36

Enzymes Localization Within the Cell  Enzymes have fixed locations within the cell  Act as structural components of particular membranes or in solution within specific membrane– enclosed eukaryotic organelles, organelles each with its own internal chemical environment  For example, in eukaryotic cells, the enzymes for cellular respiration reside in specific locations within mitochondria 37