ENZYME BIOLOGICAL CATALYST Enzyme As Catalyst All enzymes

ENZYME- BIOLOGICAL CATALYST

Enzyme As Catalyst • All enzymes are proteins - with the exception of some RNAs that catalyze their own splicing all enzymes are proteins • In general, names end with suffix “ase” - tyrosinase (tyrosine), celullase (cellulose), protease (protein), lipase (lipid) • Enzyme: a biological catalyst • enzymes can increase the rate of a reaction by a factor of up to 1020 over an uncatalyzed reaction

• Catalysis: • Catalyst: -

Enzyme As Catalyst • Enzymes are catalysts: - increase the rate of a reaction - not consumed by the reaction - act repeatedly to increase the rate of reaction - enzymes often “specific” – promote only 1 particular reaction, others catalyze a family of similar reactions Ø cellulase – cellulose as substrate Ø hexokinase – any 6 ring monosaccharide fructose, glucose

General Properties of Enzyme • Higher reaction rates: • Milder reaction rates: • Greater reaction specificity:

Enzyme Catalysis Active site - part of enzyme to which the substrate binds and the reaction takes place Substrate – a reactant in an enzyme-catalyzed reaction Product Enzyme-substrate (ES) complex – the intermediate formed when the substrate is bind at the active site of an enzyme

Enzyme Catalysis GENERAL FORMULA E = enzyme S = substrate P = product

Enzyme catalysis reaction • Physically interact with their substrates to effect catalysis • E + S ES ES* EP E + P • Where: - E = enzyme - ES = enzyme/substrate complex - ES* = enzyme/transition state complex - EP = enzyme/product complex - P = product

Enzyme catalysis reaction • Substrate bind to the enzyme’s active site pocket in the enzyme • Catalytic site = active site = where reaction takes place

Enzyme catalysis reaction • E + S ES ES* EP E + P • 1 st step: enzyme binds to substrate molecule to form an enzyme – substrate complex • E+S ES Enzyme

Enzyme catalysis reaction • E + S ES ES* EP E + P • 2 nd step: Formation of the transition state complex where the bound substance is neither product nor reactant • ES ES*, ES≠ES

Enzyme catalysis reaction • E + S ES ES* EP E + P • 3 rd step: Formation of the enzyme – product complex ES* EP

Enzyme catalysis reaction • E + S ES ES* EP • 4 th step: Release of product EP E + P

Enzyme catalysis reaction • Enzyme can only work on one substrate molecule at a time • Not change during the reaction • One product is release, enzyme is available to accept another substrate molecule

Enzyme Catalysis • Rate of reaction = reaction velocity (V) - the rate of enzyme reaction is measured by the rate of the appearance of products or the rate of disappearance of substrates. - d[P]/d. T or d[S]/d. T mol product/min or mol substrate/min • Enzyme activity? Ø 1 unit (U) is the amount of enzyme that catalyses the reaction of 1 mol of substrate per minute under specified conditions.

Enzyme Catalysis • The rate of a reaction depends on its activation energy, DG°‡ • an enzyme provides an alternative pathway with a lower activation energy • Activation energy – the energy required to start a reaction • Transition state – the intermediate stage in a reaction in which the old bonds break and new bonds are formed

How enzyme work? • Transition state theory: – The enzyme (E) must approach the substrate (S), the substrate attach to the active site through noncovalent bond – Formed the high energy (unstable) ES complex – In ES complex, the covalent bond in substrate is in the process of breaking while the EP complex is forming.

Enzyme Catalysis - Example • Consider the reaction catalase a) No catalyst, b) with added Fe 3+ salt, c) with added catalase

• (a) – a/e for the reaction in the absence of a catalyst • (b) – a/e lowered in the presence of an iron catalyst • (c) – energy diagram for the catalasecatalyzed breakdown of H 2 O 2 a/e – activation energy • (d) – energy diagram for the noncatalysed breakdown of H 2 O 2 at elevated temperature

Active site • Has specificity – can discriminate among possible substrate molecules - others recognize a functional group (group specificity) - only recognize one type of molecule (eg. D vs L isomer) (absolute specificity) • Relatively small 3 D region within the enzyme - small cleft or crevice on a large protein • Substrates bind in active site by weak non-covalent interactions (Hydrogen bond, hydrophobic and ionic interaction)

Active site • The interactions hold the substrate in the proper orientation for most effective catalysis • The energy derived from these interactions – binding energy • Binding energy is used, in large part to lower the activation energy and stabilize the transition state complex (ES*) • Each non-covalent interaction provides energy to stabilize the transition state

Binding Models • Two models have been developed to describe formation of the enzyme-substrate complex 1. Lock-and-key model: substrate binds to that portion of the enzyme with a complementary shape 2. Induced fit model: binding of the substrate induces a change in the conformation of the enzyme that results in a complementary fit

Enzyme/substrate interaction 1. Lock and key model - substrate (key) fits into a perfectly shaped space in the enzyme (lock) - lots of similarities between the shape of the enzyme and the shape of the substrate - highly stereospecific - implies a very RIGID inflexible active site - site is preformed and RIGID

Enzyme/substrate interaction 2. Induced fit model (hand in glove analogy) - count the flexibility of proteins - substrate fits into a general shape in the enzyme, causing the enzyme to change shape (conformation); close but not perfect fit of E + S - change in protein configuration leads to a near perfect fit of substrate with enzyme Figure 6. 3, pg 148 Campbell & Farrell, Biochemistry, 6 th Ed. , 2009, Thomson Brooks/Cole

Key words for today • You need to understand: 1. Factors effecting enzyme reaction rate 2. 6 classes of enzymes 3. Cofactor, coenzymes, holoenzymes, apoenzymes 4. Allosteric enzyme, effectors (positive and negative), heterotropic and homotropic allosterism 5. Isoenzyme and multienzyme

Characteristics of enzyme reactions • What influence the enzyme reaction rate? 1. Substrate concentration 2. Temperature 3. p. H 4. Enzyme concentration 5. Inhibitor

Characteristics of enzyme reactions • Substrate Saturation: [substrate] = substrate concentration

Characteristics of enzyme reactions • Temperature

Characteristics of enzyme reactions • Effects of Temperature: Ø All enzymes work within a range of temperature specific to the organism. Ø Increases in temperature lead to increases in reaction rates - is a limit to the increase because higher temperatures lead to a sharp decrease in reaction rates - due to the denaturating (alteration) of protein structure resulting from the breakdown of the weak ionic and hydrogen bonding that stabilize three dimensional structure of the enzyme.

Characteristics of enzyme reactions • p. H pepsin in the stomach works best at a p. H of 2 and trypsin at a p. H of 8.

Characteristics of enzyme reactions • Effects of p. H: Most enzymes are sensitive to p. H and have specific ranges of activity.

Characteristics of enzyme reactions • Enzyme concentration

Characteristics of enzyme reactions • Inhibitor

Classification of Enzymes • Have 6 categories • Each enzyme has an official international name ending with –ase and a classification number • Number consists in 4 digits (referred to a class and subclass of reaction

Classification of Enzymes

Classification of Enzymes Table 5. 1, pg 136 Boyer, R. , Concepts in Biochemistry, 3 rd Ed. , 2006, John Wiley &Sons

Enzyme Classes: Examples Class Example 1 (oxidoreductase) alcohol dehydrogenase 2 (transferase) hexokinase 3 (hydrolase) chymotrypsin Reaction glucose + ATP glucose-6 -phosphate + ADP polypeptide + H 2 O peptides More examples: Refer Table 5. 2, pg 136 , Boyer, R. , Concepts in Biochemistry, 3 rd Ed. , 2006, John Wiley &Sons

Enzyme Classes: Examples Class Example 4 (lyase) pyruvate decarboxylase 5 (isomerases) alanine racemase 6 (ligases) pyruvate carboxylase Reaction D-alanine L-alanine

Enzymes & cofactor • Enzymes require chemical entity in order to function properly (assists an enzyme in catalytic action) • Cofactor – nonprotein molecule that assist in an enzyme catalytic reaction • Coenzyme – smaller organic or organometallic molecule derived from vitamin, weakly bound to enzyme, temporarily associated with enzymes • Prosthetic group – coenzymes that are covalently or noncovalently tightly bound to enzyme and always present.

Enzymes & cofactor • Holoenzyme – an enzyme in its complete form including polypeptide(s) and cofactor • Apoenzyme – enzyme in its polypeptide form without any necessary prosthetic groups or cofactors

Allosteric Enzymes • Allosteric enzyme: an oligomer whose biological activity is affected by other substances binding to it • these substances change the enzyme’s activity by altering the conformation(s) of its 4°structure • Allosteric effector: a substance that modifies the behavior of an allosteric enzyme; may be an • allosteric inhibitor = negative effectors • allosteric activator = positive effectors

Allosteric enzyme in feedback Inhibition Formation of product inhibits continued production – feedback inhibition

Allosteric Enzymes (Cont’d) • The key to allosteric behavior is the existence of multiple forms for the 4°structure of the enzyme • allosteric effector: a substance that modifies the 4° structure of an allosteric enzyme • homotropic effects: allosteric interactions that occur when several identical molecules are bound to the protein; e. g. , the binding of aspartate to ATCase • heterotropic effects: allosteric interactions that occur when different substances are bound to the protein; e. g. , inhibition of ATCase by CTP and activation by ATP ATCase = aspartate transcarbomylase CTP = cytidine triphosphate

Allosteric enzymes • • • A change in conformational structure at one location of a multisubunit protein that causes a conformational change at another location on the protein Effectors – i) serves as stimulants to enzyme (+ve effectors) = increase catalytic activity – ii) inhibitors (-ve effectors) to enzyme = reduce/inhibit catalytic activity - Act by reversible, noncovalent binding to a site on the enzyme Larger and more complex than nonallosteric enzyme Have 2 or more subunits (oligomeric) Allosteric enzymes have regulatory sites for binding of substrates and reaction (catalytic sites)

HOMOTROPIC ALLOSTERISM • Eg. Tetrameric allosteric enzyme composed of 4 identical subunits • Each subunit has a catalytic site where substrate/effector will bound and transformed to product • Once bound to active site, a message will transmitted via conformational changes to an active site on another subunit which makes it easier for a substrate molecule to bind and react at that site • This type (substrate and effector) are the same is called cooperative or homotropic

HETEROTROPIC ALLOSTERISM • A dimer with nonidentical subunits • Subunit α contain the active site – catalytic subunit • Subunit β contains the site for effector binding – regulatory subunit • Binding of a specific effector molecule to the regulatory site on the β subunit sends a signal via conformational changes to the catalytic site on subunit α • Substrate and effector different kinds of molecules - heterotropic

Isoenzymes • Enzymes that catalyze the same reaction (catalytically and structurally similar) but are encoded by different genes – Glycogen phosphorylase-synthesize in liver, brain and muscle-involves in degradation of glycogen – Isoenzymes = isoforms

Multienzymes – A group of noncovalently associated enzymes that catalyze 2 or more sequential steps in metabolic/biochemical pathway