enzymes

reactions that occur in a cell

• chemical reactions that occur in cells constitute the process of metabolism

  • metabolism is a basic characteristic of all living system → metabolic reactions keep the organism alive

• two types of metabolic reactions occur within the cell: anabolic and catabolic reaction ⇒ controlled by enzymes

1. anabolic reactions

   • synthesis of complex compounds from simple molecules

   • require energy

   • form complex biomolecules required for cell structures and energy storage

   • e.g.: synthesis of starch, glycogen, lipids and proteins

2. catabolic reactions

   • breakdown of complex compounds into simple molecules

   • release energy

   • break down complex biomolecules, releasing energy for ATP synthesis

   • e.g.: for mobilising food stores, making energy available for cells

pathway of metabolism

• participating molecules in metabolic reactions → metabolites

  • not converted into products in single, large reactions

  • are converted gradually through a series of reactions which constitute a metabolic pathway

• reasons why metabolism proceeds in small steps

  1. many catabolic reactions create unfavourable conditions, such as producing very high temperatures which is unsuitable for life processes

  2. energy can be derived from some catabolic reactions in a usable form

  3. substances that are partially broken down (intermediates) provide raw materials for other reactions

     • certain intermediate compounds in a catabolic pathway may have their own functions

  4. under normal conditions in the cell, it is impossible to synthesise complex organic compounds from simple raw materials in one step

  5. having small steps in a metabolic pathway allows the cell to better control the products made

enzymes → definition

enzymes are protein molecules which greatly increase the rate of a chemical reaction without themselves being changed at the end of the reaction

enzymes

• enzymes are specific in the reactions they catalyse

  • a single enzyme generally catalyses only a single reaction

• without enzymes, biochemical reactions will proceed too slowly to sustain life

  • raising temperature can increase the speed of reaction, but this is detrimental to the cell

• enzymes enable metabolic reactions to proceed rapidly at low temperatures

• enzymes allow the cell to control the metabolic pathways in the cell

intracellular enzymes

• enzymes that function within the cell

extracellular enzymes

• enzymes produced in the cells but are packaged to be secreted → work externally

• e.g.: most digestive enzymes

naming and classification of enzymes

• substrate: molecules in which the enzyme is acting upon

• enzyme: named by attaching the suffix ‘-ase’ to the name of the substrate on which it acts

  • e.g.: enzymes that act on carbohydrates: carbohydrases; enzymes that act on proteins: proteases

• 6 categories based on type of chemical reaction: oxidoreductase, transferase, hydrolase, lyase, isomerase, ligase

general structure of enzymes

• enzymes are protein in nature

• all enzymes have a three-dimensional globular shape (tertiary structure) and are relatively large molecules → large and complex

• however, only a small portion of the enzyme molecule comes into direct contact with the substrate ⇒ active site, and binding with the substrate occurs here

characteristics of the active site

• has a shape which is complementary to the shape of the substrate

• two types of residues are found in the active site → catalytic and contact residues

  1. catalytic residues

     • directly act on the bonds in the substrate which are broken/formed by enzyme action

     • are responsible for the ability of the enzyme to catalyse chemical reactions

  2. contact residues

     • responsible for the specificity of the enzyme

     • ensures/make shape of the active site complementary to the shape of the substrate

• remaining amino acid residues(structural residues), forms the bulk of the enzyme which function to maintain the globular shape of the enzyme ⇒ essential for the optimal function of the active site

activation energy

activation energy is the amount of energy that reactants must absorb before they can react to form products (required to make substances react)

• represents the energy barrier that has to be overcome before a reaction can take place to form products

• the greater the activation energy, the slower will the reaction occur at any particular temperature

• if the activation energy of a reaction is lowered, rate of reaction would be increased → lower energy barrier

how enzymes work

• activation energy can be supplied in the form of heat energy ⇒ allows more reactants to react to form products per unit time → not possible in the living cell

• cells only survive within a narrow range of relatively low temperature → an increase in the temperature will often kill the cell

• enzymes are special biological catalysts, which serve to reduce the activation energy required for the reaction + speed up the overall rate of reaction without altering the temperature at which the reaction occurs

process of how enzymes work

1. effective collision between substrate and enzyme at the correct orientation causes substrate to bind to the enzyme molecule at its active site, to form a short-lived enzyme-substrate complex, and the chances of successful reactions occurring are greatly enhanced in the complex

2. the substrate molecules react together

3. once the reaction has occured, the products dissociate from the complex, and the unchanged/unaltered enzyme molecule is also released, and is then available to catalyse another cycle of reaction

   • shape of products are not complementary to the shape of the active site

how do enzymes lower the activation energy of a reaction

• active site serves as a platform for substrates to collide at the correct orientation for chemical reactions to occur ⇒ products are formed quickly

  • bonds are formed at specific places

• enzyme-substrate complex distorts the bonds in the substrate

  • in active site, certain bonds in the substrate molecule may be placed under physical stress

  • increases the likelihood that the bond will break (specific bonds break easily → products are formed quickly)

• the catalytic amino acids at the active site changes substrate reactivity

  • R-groups on catalytic amino acids of enzymes (come close to substitutes):

    • change the charge of the substrate

    • alter distribution of electrons within bonds of substrate

    • cause other chemical changes which increase reactivity of substrate

mechanism of enzyme action

• enzymes are highly specific in the reactions they catalyse

  • some enzymes catalyse the transformation of one particular type of substrate, or at the most, a very restricted group of substrates → absolute specificity

  • some enzymes catalyse only on molecules that have specific functional groups such as amino, phosphate and methylene groups → group specificity

• two hypothesis: lock-and-key hypothesis and induced fit hypothesis

lock-and-key hypothesis

• the active site has a specific shape, to which the substrate binds

• substrate (key) has a shape complementary to shape of the active site (lock)

• the shape of the substrate fits exactly into the shape of the active site

• once the reaction is completed, products no longer fit into active site, and are released, leaving the active site free to receive new substrate molecules

induced fit hypothesis

• stemmed from evidence that suggested that some enzymes and their active sites were physically flexible structures

• shape of substrate is still complementary to the shape of the active site but does not fit exactly

• binding of substrate of active site induces a small conformational change in the shape of the enzyme

  • this enables the substrate to fit more snugly into the active site to form the enzyme-substrate complex

  • this allows the enzyme to perform its catalytic function more effectively

differences between lock-and-key and induced fit hypothesis

point of comparison	lock-and-key	induced fit
shape of active site and substrate	shape of substrate is exactly complementary to the shape of the active site	shape of active site is complementary but substrate does not fit snugly
conformational change of enzyme upon substrate binding	does not cause conformational change to the enzyme after substrate binds	substrate induces a slight conformational change in the shape of the enzyme to fit more snugly into the active sites

properties of enzymes

• most enzymes are globular proteins → each enzyme molecule has an active sites where the reaction takes place, and the active site has a shape that is complementary to the shape of the substrate

• enzymes function as biological catalysts and hence share the properties of catalysts

  • effective in small amounts

  • remain chemically unaltered at the end of the reaction they catalyse

  • lower the activation energy required for the reaction to occur

• enzymes are extremely efficient

  • many enzyme-catalysed reactions proceed 10³ to 10⁸ times faster than uncatalysed reactions

  • enzymes have a high turnover number → number of moles of substrate converted by one mole of enzyme per minute ⇒ reflection of the speed of enzyme action

• enzymes have a high degree of specificity

  • most enzymes are specific to a particular substrate molecule, while other enzymes are specific to a group of closely related substrates or catalyse a specific type of reaction

  • specificity of an enzyme is due to the conformation of its active sites

    • R group of catalytic residues form attractions with substrates (affinity)

  • only substrate whose shape is complementary to the shape of active site can bind to the enzyme → enzyme-catalysed reaction

• activity of enzymes is affected by changes in pH, temperature, substrate concentration and enzyme concentration

• activity of enzymes can be altered by presence of inhibitors (slow down) or activators (speed up)

  • implies that the rate of product formation can be controlled according to the needs of the cell

• enzyme catalyse reactions that are usually reversible

  • reversible reactions proceed in a bi-directional manner until an equilibrium is reached

  • enzymes speed up the rate at which equilibrium is reached

temperature → factors affecting rate of enzyme action

• as temperature increases, there is an increase in kinetic energy of enzyme and substrate molecules

• this results in an increase in number of effective collisions between enzyme and substrate molecules

  • more enzyme-substrate complexes are formed per unit time → higher rate of formation

• rate of enzyme catalysed reaction is doubled for every 10° rise in temperature before optimum temperature is reached

  • the effect of temperature on the rate of reaction can be expressed as the temperature coefficient, Q₁₀

    • Q₁₀ = rate of reaction (x + 10) °C/rate of reaction at x °C

  • for most enzymes, the Q₁₀ value is approximately 2 → provided optimum temperature is not reached

• the rate of reaction increases with temperature until the optimum temperature is reached

• if the temperature is increased beyond the optimum temperature, the rate of reaction decreases steeply

  • as temperature increases, there is an increase in kinetic energy supplied to the enzyme

  • this causes atoms in the enzyme to vibrate more, resulting in breaking of hydrogen bonds and hydrophobic interactions between R-groups of amino acid residues which stabilise the tertiary and quaternary structures of enzyme

  • enzyme unfolds and the precise shape of the active site is lost → denatured

  • enzyme denaturation is usually irreversible

• if the temperature is reduced to near or below freezing point, the enzymes are inactivated

  • enzyme activity is very low, but they will regain their catalytic influence when higher temperatures are restored

optimum temperature

optimum temperature is the temperature at which the enzyme is functioning at its maximum rate

• optimum temperature of most mammalian enzymes lie between 30 - 40 °C, but enzymes with higher optimum temperatures exist

to explain effects of temperature

1. as temperature increases, there is an increase in kinetic energy of enzyme and substrate molecules

   • an increase in number of effective collisions between enzyme and substrate molecules

   • more enzyme-substrate complexes formed per unit time

   • rate of reaction increased

2. rate of reaction was highest at __, which is the optimal temperature of the enzyme

3. beyond this temperature, enzymes are denatured → intramolecular bonds between R groups of amino acid residues are broken ⇒ rate of reaction decreased with further increase in temperature

effects of pH → factors affecting rate of enzyme action

• under conditions of constant temperature, every enzyme functions over a particular range of pH

• at optimum pH, the intramolecular bonds which maintain the tertiary structure of the enzyme are intact

  • the conformation of the active site is most ideal for substrate binding

  • the frequency of effective collisions between enzyme and substrate molecules is the highest, and hence there is the greatest number of enzyme-substrate complexes formed per unit time

• for pH higher or lower than optimum pH, rate of reaction decreases

  • at pH higher or lower than optimum pH, the H+ concentration is changed

  • this alters ionic charges on the basic and acidic r-groups of amino acid residues on enzyme molecule

  • ionic bonds (and maybe hydrogen bonds) are disrupted and substrate binding is affected

  • shape of active site is changed and is less complementary to shape of substrate

  • rate of effective collisions decreases and less enzyme-substrate complexes formed per formed per unit time → less products formed

• at this small range of pH, the effects of pH are normally reversible → restoring the pH to the optimum would usually restore the optimum rate of reaction

• at extreme pH (further away from optimum pH), the rate decreases further

  • the enzyme is denatured → the conformation of active site is no longer complementary to shape of substrate

  • less frequency of effective collisions between enzyme and substrate molecules and fewer number of enzyme-substrate complexes formed per unit time ⇒ lesser products are formed

optimum pH

the pH at which maximum rate of reaction occurs

effects of substrate concentration → factors affecting rate of enzyme action

for fixed enzyme concentration

• at low substrate concentration, the rate increases steeply with increasing substrate concentration

• increase in number of substrate molecules lead to increase in frequency of effective collisions between enzyme and substrate

  • more enzyme-substrate complexes formed per unit time

  • rate is increased

• rate of reaction is limited by substrate concentration

• at high substrate concentration, rate remains the same even with further increase in substrate concentration → graph plateau

  • this is because all active sites of enzymes are saturated with substrate molecules

  • extra free substrate molecule has to wait until product is released before it can enter active site of enzyme

• rate of reaction is now limited by enzyme concentration

affinity of enzymes for its substrate

• reflected by the michaelis constant K_m → concentration of substrate required for reactions to proceed at half of its maximum rate

• K_m is always the same for a particular enzyme, but varies from one enzyme to the other

• low K_m → there is high affinity between enzyme and substrate (less substrate is needed to achieve 1/2 Vmax)

• high K_m → there is low affinity between the enzyme and substrate (more substrate is needed to achieve 1/2 Vmax)

effects of enzyme concentration

• if substrate concentration is maintained at a high level, and other conditions such as pH and temperature are kept constant, the rate of reaction will be proportional to the enzyme concentration

  • as enzyme concentration increases, frequency of effective collisions between enzyme and substrate molecules increases

    • more enzyme-substrate complexes formed per unit time

    • greater rate of product formation

• at very high enzyme concentrations, if the concentration of substrate molecules is limiting, an increase in enzyme concentration would not result in any further increase in the rate of reaction → rate of enzymatic reaction is said to be limited by substrate concentration

enzyme inhibitors

inhibitors reduce or stop the rate of reaction

• inhibitor: a substance which prevents an enzyme from catalysing its reaction

• they combine with the enzyme to form enzyme-inhibitor complexes so that substrate molecules cannot bind to the enzyme

• if inhibitor can dissociate, the inhibition is reversible

  • these inhibitors form a relatively loose association with the enzyme

  • can be removed from the enzyme under certain conditions

• if inhibitor cannot dissociate, then the inhibition is irreversible

  • these inhibitors bind permanently to the enzyme

  • very low concentration of inhibitors are sufficient to completely inhibit some enzymes

competitive inhibitors

• inhibitors are structurally similar to the substrate of the enzyme

• competitive inhibitors compete with substrate molecules for the active site of the enzyme

  • shape of inhibitor is complementary to the shape of (part of) active site

• inhibitor blocks active site and substrate cannot bind

• effect of a competitive inhibitors can be reduced by increasing the concentration of substrate

  • this increases the probability of an enzyme-substrate complexes formation rather than enzyme-inhibitor complexes formation

  • rate of reaction can thus be increased

• e.g.: penicillin blocks the active site of the enzyme transpeptidase, which is used by bacteria to construct the cell wall

non-competitive inhibitors

• have no structural resemblance to the substrate

• binds permanently to the enzyme in a region other than its active site

  • results in a change in the conformation of the enzyme molecule, including the conformation of its active site

  • the substrate is unable to bind to the active site of the enzyme

  • enzyme-substrate complex cannot form

• non-competitive inhibitor puts a proportion of the enzyme molecules out of action

  • the effective enzyme concentration is lowered

• reaction rate cannot reach its maximum, even when substrate concentration is increased

• e.g.: cyanide is a poison which prevents ATP production via aerobic respiration → binds to a region away from the active site on cytochrome oxidase, an enzyme that forms part of the electron transport chain

differences between competitive and non-competitive inhibition

	competitive inhibition	non-competitive inhibition
site of binding of inhibitor	at the active site of the enzyme → competes with substrate	at a region other than the active site of the enzyme
structure of inhibitor	structally similar to the substrate	no structural resemblance with the substrate
effect of increasing substrate concentration on inhibition reaction rate	effects of inhibitor are not observed	inhibitory effects at still observed
maximum rate of reaction	attained if substrate concentration are sufficiently high	never attained regardless of how high the substrate concentration is

allosteric interactions

• some enzymes are regulated by allosteric compounds which bind to these enzymes at specific sites away from the active site

  • enzymes are known as allosteric enzymes (has a quaternary structure - more than 1 subunit)

• compounds modify enzyme activity by causing a conformational change in the structure of the enzyme’s active site

  • affects the ability of the substrate to bind to the enzyme

allosteric inhibitors

• binds to specific sites away from enzyme active site, causing a change in the conformation of the enzyme’s active site

• substrate is unable to bind to active site and results in a reduced rate of reaction

• does not bind permanently

allosteric activators

• binds to specific sites away from enzyme active site, causing a change in the conformation of the enzyme’s active site

• substrate binds well to active site, and results in an increased rate of reaction

end-product inhibition

• a metabolic pathway usually involves many intermediate reactions, each controlled by an enzyme

• when the end-product of a metabolic pathway begins to accumulate, it may act as an inhibitor, usually on the enzyme controlling the first step in the pathway → the product is able to switch off its own production as it builds up

• the process is self-regulatory → as product is used up, the inhibition is lifted and the production is switched back on again

enzyme cofactors

• most enzymes require non-protein components in order to function → cofactors (increase function and efficiency)

  • vary from simple inorganic ions to complex organic molecules

inorganic ions → enzyme activators

• either change shape of the enzyme or shape of substrate to facilitate the formation of enzyme-substrate complex, increasing the rate of an enzyme-catalysed reaction

• e.g.: salivary amylase activity is increased in the presence of chloride ions

prosthetic groups

• are cofactors which are tightly bound to the enzyme on a permanent basis

• are organic molecules

• assist in the catalytic function of the enzyme → function to transfer atoms or chemical groups from the active site of the enzyme to some other substance

• e.g.: catalase has an iron-containing haem prosthetic group

coenzymes

• organic molecules which act as cofactors

• do not remain attached to the enzyme between reactions → only loosely associated with the enzyme during the reaction

• function as carriers that transfer chemical groups or atoms from the active site of one enzyme to the active site of another enzyme

• all coenzymes are derived from vitamins

• e.g.: nicotinamide adenine dinucleotide is derived from the vitamin nicotinic acid, and is an important coenzyme in respiration