CHAPTER 4: BIOCATALYSIS 1. PROPERTIES OF ENZYME & MECHANISM OF ACTIONS 2. COFACTOR 3. INHIBITORS BIOLOGY 2 SB025 SEMESTER 2 SESSION 2022/2023 CHAPTER 4: BIOCATALYSIS
BIOLOGY 2 SB025 SEMESTER 2 SESSION 2022/2023 CHAPTER 4: BIOCATALYSIS 1. PROPERTIES OF ENZYMES & MECHANISM OF ACTIONS LEARNING OUTCOMES a) Describe the structure of enzyme as globular protein that can catalyse metabolic reaction. b) Describe the six classes of enzyme with example according to IUBMB classification. c) Describe the factors that affect the enzymatic reaction : substrate concentration, pH, temperature and enzyme concentration.
Macromolecule that acts as catalyst, a chemical agent that speeds up a reaction without being consumed by the reaction.
• Enzyme is globular protein and has tertiary @ quaternary structure. • An enzyme has active site. Active site is the specific site (region) of an enzyme that binds to specific substrate in which catalysis occur. • Somes enzyme have an allosteric site (other site than active site on enzyme) STRUCTURE OF AN ENZYME (3-D configurations of an enzyme)
ENZYME METABOLIC REACTION • An enzyme has active site. Active site is the specific site (region) of an enzyme that binds to specific substrate in which catalysis occur. • The specificity of an enzyme is due to the complementary shape of its active site and the shape of the substrate. • When an enzyme is denatured by heat or a change in pH, the shape of active site is changed and the substrate can no longer fit into active site.
• Substrate binds at active site of enzymes forming enzyme-substrate complex • Enzymes facilitate the breaking of old bond between substrate and formation of new bonds between product. • After the reaction, product form and enzyme can be reusable. ENZYME METABOLIC REACTION
SUMMARY OF ENZYME REACTION E + S S P E E + Enzyme Substrate (Reactant) Enzyme-Substrate Complex (transition state) Enzyme Product
⮚ Recommended by International Union of Biochemistry and Molecular Biology (IUBMB) based on the type of reaction the enzyme catalyses. ⮚ Naming by adding suffix-ase :- 1. + + 2. + CLASSIFICATION OF ENZYMES Type of reaction it catalyse Substrate Suffix ase Name of substrate Suffix ase
6 CLASS OF ENZYME 2. Transferases 3. Hydrolases 5. Isomerases 1. Oxidoreductases 4. Lyases 6. Ligases CLASSIFICATION OF ENZYMES
OXIDOREDUCTASES - Catalyse oxidation-reduction reactions - Need coenzymes FAD, NAD+ , NADP+ Example : dehydrogenase, oxidase, oxygenase, peroxidase, catalase
MALATE OXALOACETATE Malate dehydrogenase NAD+ NADH + H+ • Malate is oxidized to oxaloacetate • NAD+ is reduced to NADH + H+ Example: Malate dehydrogenase OXIDOREDUCTASES
SUCC IN IC AC ID FUMAR IC AC ID Succinic dehydrogenase FAD FADH2 • Succinic acid is oxidized to fumaric acid • FAD is reduced to FADH2 Example: Succinic dehydrogenase OXIDOREDUCTASES
- Catalyse the transfers of a functional group (e.g. methyl, amino, phosphate) from a donor molecule to an acceptor molecule. - Example : phosphorylase, transaminase, kinase TRANSFERASES
G lucose Glucose-6-phosphate hexokinase Example: Hexokinase TRANSFERASES ATP ADP Hexokinase - catalyse the transfer of phosphate group from ATP to glucose
- Catalyse hydrolysis reactions - Example : Digestive enzyme : protease, lipase, amylase, sucrase, lysozyme sucrose H fructose + α-glucose + 2O HYDROLASES sucrase
- Catalyse certain reactions in which double bond form or break - Example : decarboxylase, carboxylase, aldolase LYASES
Pyruvate ethanal Pyruvate decarboxylase CO2 Pyruvate decarboxylase - catalyse removal of CO2 from pyruvate Example: Pyruvate decarboxylase LYASES
PEP oxaloacetate PEP carboxylase Example: PEP carboxylase LYASES CO2 PEP carboxylase : catalyse addition of CO2
- Catalyse conversion of a molecule from one isomeric form to another. - Example: Isomerase, Fumarase glucose -1- phosphate phosphoglucomutase glucose -6- phosphate ISOMERASES
AminoacyltRNA Amino synthetase acid + tRNA - Catalyse certain reactions in which two molecules become joined in a process coupled to the hydrolysis of ATP. Aminoacyl-tRNA + ATP AMP 2 Pi LIGASES
FACTORS AFFECT THE ENZYMATIC REACTION 1.SUBSTRATE CONCENTRATION 2. pH 3. TEMPERATURE 4.ENZYME CONCENTRATION
EFFECTS OF SUBSTRATE CONCENTRATION • At low substrate concentration, rate of enzyme reaction increases with increasing substrate concentration • Not all active sites bind with substrates(some active site remain free). The active site of an enzyme molecule can bind with a certain number of substrate molecules at a given time.
• As substrate concentration increase,the rate of reaction increase - collision between substrates& active sites of enzymes also increase - more active sites bind with substrates -Untilthere are no more free active sites // saturation of active site EFFECTS OF SUBSTRATE CONCENTRATION
• At very high substrate concentration, reaction rate become constant • Any further increase in substrate concentration will not increase rate of reaction • Because all active sites are bind with substrates/ enzyme active site isfully saturated with substrate EFFECTS OF SUBSTRATE CONCENTRATION
EFFECTS OF pH • Enzymes function efficiently within narrow pH range • Different enzymes have different optimum pH ✔ e.g: pepsin=pH2 ,trypsin=pH8 • Optimum pH: • pH at which reaction rate ismaximum • Above or below optimum pH,the rate of reaction decrease
EFFECTS OF pH • Deviation from optimum pH range results in excessH+ or OH- • that alters the acidic / basic/ functional group / side chain of amino acids in the enzyme • Causing ionic or hydrogen bonds to be broken • Change conformation of the active site // enzyme denatured • Substrate cannot bind to active site • Lower down the rate of reaction
EFFECTS OF pH • IfpH istoo low or extreme, enzyme will denature - Result from the breaking of ionic bonds - Substrate can’t bind to active site of enzyme
• At low temperature,the rate of reaction occur slowly ✔ due to the slow movement of molecules -collision between substrate & active site are decrease ✔ enzyme also inactive at low temperature EFFECTS OF TEMPERATURE Rate of Reaction Temperature 0C
• As temperature increase,the rate of reaction increase ✔ movement of molecules increase ✔ collision between substrate & active sites of enzymes are increase (more frequent) EFFECTS OF TEMPERATURE Temperature 0C Rate of Reaction
EFFECTS OF TEMPERATURE • For every 10ºC rise in temperature, reaction rate is doubled • up to optimum temperature at which reaction rate ismaximum • Optimum temperature: ✔Human enzymes mostly at 350C – 400C (37.5°C) ✔Enzyme of thermophilic bacteria (live in hot spring) is700C
• Higher temperature (beyond optimum temperature) , reaction rate decrease ✔ >40°C: activity decrease ✔at 60°C: reaction stops&enzyme is denatured • Due to denaturation of protein ✔ changes in conformation of active site ✔ Results from the breaking of ionic bond / hydrogen bond /disulphide bridge / hydrophobic &van der Waals interactions • Substrate can no longer fit into the active site of the enzymes EFFECTS OF TEMPERATURE
EFFECTS OF TEMPERATURE Temperature 0C Rate of Reaction
EFFECTSOF ENZYME CONCENTRATION • The rate of reaction is directly proportional to enzyme concentration with the condition the pH and the temperature are kept constant at optimum. • And with the presence of excessive substrate • The shape of the graph islinear
BIOLOGY 2 SB025 SEMESTER 2 SESSION 2022/2023 CHAPTER 4: BIOCATALYSIS 4.2 COFACTOR LEARNING OUTCOMES a) Define cofactor
DEFINITION A non-protein molecule or ion that is required for proper functioning of an enzyme. COFACTOR • It can be permanently bound to the active site or may bind loosely and reversibly with the substrate during catalysis.
Apoenzyme : an inactive enzyme without cofactor. Holoenzyme: an apoenzyme together with cofactor. COFACTOR
TYPES OF COFACTOR Inorganic Organic loosely & temporarily to enzyme active site Loosely & temporarily to enzyme active site Tightly & permanently to enzyme active site Coenzyme Prosthetic group Metal ion activator e.g: Zn2+ , Mg2+ e.g: NAD+ , NADP+ e.g: Haem
4.3 INHIBITORS LEARNING OUTCOMES a) Define inhibitor b) State the types of inhibitors: i. competitive inhibitors ii. non-competitive inhibitors BIOLOGY 2 SB025 SEMESTER 2 SESSION 2022/2023 CHAPTER 4: BIOCATALYSIS
INHIBITOR COMPETITIVE INHIBITORS NON-COMPETITIVE INHIBITORS Molecules that prevent catalyzing reactions when they are bound to the enzymes. • Similar shape with substrate • Compete for same active site • Example: Malonate • Not similar shape with substrate • NOT compete for same active site • Example: Isoleucine allosteric site
EFFECT OF INHIBITORS • Will decreases the rate of enzymecontrolled reactions. • Removal of the inhibitor, restore the activity of enzyme to normal • E.g. malonate (malonic acid), isoleucine, ATP
• Similar shape with substrate • Compete for same active site • When the inhibitor bind to active site; - prevent substrate from bind to active site - enzyme-substrate complex do not form - product do not form - reduce/slow down rate of reaction COMPETITIVE INHIBITORS
COMPETITIVE INHIBITORS
Succinic acid is substrate for succinic dehydrogenase Malonic acid act as inhibitor COMPETITIVE INHIBITORS • Example of competitive inhibitor : Malonate (malonic acid)
How the activity of enzyme can be optimized to less the effect of inhibition? - increase concentration of substrates - if concentration of substrates is high, substrate have greater chance to bind with active site COMPETITIVE INHIBITORS
• Different shape to the substrate/ do NOT have similar shape with substrate • Do NOT compete for same active site • The inhibitor bind to allosteric site • Cause conformational change of active site • substrate cannot bind to active site • enzyme-substrate complex do not form NON-COMPETITIVE INHIBITORS
NON-COMPETITIVE INHIBITORS
• Product do not form • Reduce rate of reaction • Increase in substrate concentration do NOT affect the reaction rate - because non-competitive inhibitor & substrate do not compete for same active site • Example of non-competitive inhibitor: Isoleucine NON-COMPETITIVE INHIBITORS
How the activity of enzyme can be optimized to less the effect of inhibition? - by removing the inhibitor - decrease concentration of the inhibitors NON-COMPETITIVE INHIBITORS
Competitive inhibitor Non-competitive inhibitor ✔ Inhibitor bind at ACTIVE SITE of enzyme ✔ Inhibitor has similar shape to substrate ✔ Inhibitor bind at ALLOSTERIC SITE of enzyme ✔ Inhibitor has different shape with substrate ✔ Inhibitor compete with substrate for same active site of enzyme ✔ Inhibitor DOES NOT compete with substrate for active site of enzyme but change active site conformation DIFFERENCES BETWEEN COMPETITIVE & NON-COMPETITIVE INHIBITOR
THANK YOU! Enzymes are substance that makes life possible. They are needed for every chemical reaction that takes place in the human body. No minerals, vitamins or hormones can do work without enzymes. Our bodies, all our organs, tissues and cells are run by metabolic enzymes. ~Dr. Howell~