DR. S.CHAKRAVARTY MD
Enzymes
Learning objectives
Define and classify enzymes based on the IUPAC agreement. Give examples to each class
Classify cofactors and give examples for various types
Discuss the general properties of enzymes and list the important functions
Define KM, Vmax, Tranistion state and activation energy of enzymes
Discusss the factors affecting enzyme activity
Explain the Michaelis Menten reaction and differentiate it from lineweaver burk plot. Classify various types of enzyme inhibitions
Definition
Thermolabile biocatalysts which enhances a chemical reaction without undergoing any chemical change.
Properties :1. Specific for a reaction2. Does not dictate the direction of a reaction3. Increases the rate of a reaction by several
thousand times4. Are proteins – except ribozymes (RNA)5. Lowers the activation energy of a reaction
Enzyme terminology
Simple enzyme – made only of proteins
Complex enzyme – also called Holoenzyme 1. Apoenzyme – protein part2. Non-protein part -
Prosthetic group –with covalent interaction ( usually metals)
co-enzyme – without covalent interaction (vitamins )
Co-enzymes
Group I: take part in reactions transferring hydrogen atoms or electrons
VITAMIN COENZYME FUNCTION
Riboflavin FMNFAD
Redoxreactions
Niacin NADNADP+
Redoxreactions
Co-enzymes
Group II: take part in reactions transferring groups other than hydrogen
Vitamin Coenzyme Group transferred
Function
Thiamine TPP Hydroxy ethyl Transketolase,oxidative decarboxylation
Pyridoxine PLP Amino or keto Transamination reaction
Folic acid FH4 One carbon group
One carbon metabolism
Biotin Biotin Carbon dioxide Carboxylation reaction
Enzymes with metals
Metalloenzyme – metal is the indegenous part of the enzyme itself. Seperation causes disruption
Metal activated enzymes – required for
activation but not indegenous to the enzyme.
Metalloenzymes
Metal Enzyme containing the metalZinc Carbonic anhydrase, Alcohol dehydrogenase,
Iron Catalase, Peroxidase, Cytochrome oxidaseXanthine oxidase
Magnesium Hexokinase, Enolase,Glucose-6-phosphatase Phospho fructo kinase
Manganese Enolase, hexokinase
Copper Tyrosinase, Lysyl oxidase
Classification of enzymes: based on function
1. Oxidoreductases
2. Transferases
3. Hydrolases
4. Lyases
5. Isomerases
6. Ligases
c. Hydroperoxidase – use hydrogen peroxide as substrate to form water
Ex: catalase, peroxidase
2H2O2 ---------- 2H2O +O2
d. Oxygenases - Monooxygenase – single atom of oxygen (hydroxylase) Ex: Phenylalanine hydroxylase
A-H+O2+ZH2 ---------- A-OH +H2O +Z
Dioxygenase – incorporate both oxygen atoms Ex: Homogentisic acid dioxygenase
A+O2 --------- AO2
2.Transferases
Transfer of groups other than hydrogen:
a. Aminotransferases : transfer of amino groups Ex: Aspartate transaminase , Alanine transaminase
b. Acyl transferases : transfer of acyl groups which requires co-enzyme A
Ex: choline acetylase
C. Methyl transferase: transfer of methyl groups Ex: Homocysteine methyl transferase
D. Phosphotransferase (kinases) – transfer of phosphate groups. Ex : Hexokinse
3.Hydrolases
Clevage of the substrates by addition of water:A. Hydrolysis of carbohydrates – maltase,
lactase, sucrase etc.
B. Hydrolysis of triglycerides – lipase
C. Hydrolysis of proteins – pepsin, trypsin, etc
D. Phosphatases – phosphodiesterase and glucose -6-phosphatase.
4.Lyases
Cleavage of substartes or removal of groups by mechanism other than addition of water.
A. Decarboxylases – removal of carbon dioxide from substrates (require B6)
Ex : histidine decarboxylase
B. Phosphorylases – clevage of substrates by addition of phosphoric acid
Ex : Glycogen phosphorylase
5.Isomerases
Catalyze intramolecular rearrangement, catalyze conversions between optical, positional and geometric isomers.
A. Aldose ketose isomerase B. Epimerase C. Racemase – interconversion of D and L formsD. Mutase – transfer of chemical group from
one position to another in th same molecule. Ex: glucose -6-po4 -- glucose-1-po4
6.Ligases
Condensation of two molecules to form one molecule using energy in the form of ATP.
A. DNA ligase
B. Synthetase/synthase Glutamic acid +ammonia -------------- Glutamine
Glutamine synthase
ATP
Catalysis occurs at the active site
Features of active site
Occupies only a small portion of the whole enzyme
Situated in a crevice or cleft of the enzyme
Possesses a substrate binding site & a Catalytic site
The substrate binds at the active site by weak noncovalent bonds
The amino acids usually found at the active site are serine, lysine, histidine, arginine, cysteine
Theories explaining the binding of substrate to the enzyme
Fischer’s template theory (lock and key model of enzyme attachment :
According to this theory the active site of the enzyme is rigid. only a specific substrate complementary to the active site fits to it just as a key fits to its proper lock
Koshland’s induced fit theory:
According to this theory the active site is not rigid & pre- shaped.
The interaction of the substrate with the enzyme induces a conformational change at the active site so that proper alignment of the catalytic residues occur & the substrate fits in the active site
E P+
+
ES
ComplexS+ E
Koshland’s Induced Fit Theory
Fischer’s template theory
E S
E S
E S+ ES
Complex E P
+
+
Mechanism of action
Lowering of activation energy
Activation energy is defined as the energy required to convert all molecules of a reacting substance from ground state to transition state
Higher the activation energy ,slower the reaction & vice versa
Activation energy
Reactant [ground state]
Product[Transition state]
A BA*
Activation energy (Ea)
Mechanism of action
Mechanism of enzyme activity
1. Catalysis by proximity
2. Acid-base catalysis
3. Catalysis by strain
4. Covalent catalysis
Catalysis by proximity
High substrate concentration –more frequent encounter and greater will be the rate.
Enzymes bind substrate at active site high local substrate concentration
Orientation of molecules for better bonding
Acid-base catalysis
The amino acids at the active site contribute to catalysis by acting as acids or bases
Histidine is often the residue involved in these acid/base reactions, since it has a pKa close to neutral pH and can therefore both accept and donate protons.
Catalysis by strain
Enzymes bind to their substrates in a conformation slightly unfavorable for the bond present in the molecule which will undergo clevage.
Resulting strain stretches the bond and breaks it.
Covalent catalysis
Involves formation of a covalent bond between the enzyme and one or more substrates
Modified enzyme becomes a reactant
Creates new reaction pathway whose activation energy is lower
Serine, histidine, cysteine are involved.
Enzyme kinetics
Quantitative measurement of rates of enzyme catalyzed reaction and the study of factors that affect the rate of reaction.
A + B P + Q
The term substrate and products in the above are arbitrary in a reaction because the reaction can take place in both directions.
The direction of the reaction is dictated by the thermodynamics (change in free energy or delta G) of the reaction to which it favours.
Bioenergetics
Also called biochemical thermodynamics
Study of the energy changes accompanying chemical reactions
Describes the transfer and utilization of energy in biologic systems
Laws of Thermodynamics:
First law of thermodynamics: The total energy of a system, including its surroundings, remains constant
Derivation:
H = Q – WEnthalpy Heat Workor Heat content absorbed done
Second Law of Thermodynamics
The total entropy of a system must increase if a process is to occur spontaneously
Entropy:
# Extent of disorder or randomness of the System
# Maximum in a system as it approaches true equilibrium
Q = T Δ S Heat Temp Entropy
Direction and equilibrium of a reaction
If G = negative i.e., free energy of product is less than free energy
of substrates, the direction of the reaction is from left to right.
these reactions are said to be spontaneous
If G = positive i.e., free energy of the product is greater than the
free energy of the substrates, the direction of the reaction is from right to left favoring substrate formation.
If G =0, then the reaction is in equilibrium.
USMLE !
Delta G is negative Delta G is positive
Actual free energy change
Spontaneous and Exergonic Non-spontaneous and Endergonic
Δ G of two consecutive reactions are additive .
As long as the sum of the Δ Gs of the individual reactions is negative, the pathway can potentially proceed to completion even if some of the individual component reactions of the pathway have a positive Δ G .
Glucose + Pi glucose 6-P + H2O ΔG° = +3.3. kcal/mol
ATP + H2O ADP + Pi ΔG° = -7.3 kcal/mol
Glucose + ATP Glucose 6-P + ADP ΔG° = -4.0 kcal/mol
Exergonic and Endergonic reactions -Coupled USMLE !
Endergonic process proceed by coupling to exergonic process
The conversion of metabolite A to metabolite B occurs with release of free energy
• Free energy is required to convert metabolite C to metabolite D
An endergonic process cannot exist independently but must be a component of a coupled exergonic-endergonic system where the overall net change is exergonic.
Factors affecting the rate of enzyme catalyzed reaction
1. Enzyme concentration
2. Substrate concentration
3. Temperature
4. pH
5. Product concentration
6. Presence of activators & Inhibitors
Enzyme concentration
1. Effect of enzyme concentration [E] on velocity of reaction [v]
As the substrate concentration is increased the velocity also correspondingly increased in the initial phases but the curve flattens afterwards.
•A rectangular hyperbola is obtained
2. Substrate concentration
Substrate concentration
½ Vmax
Km
Vmax
First order reaction
At low substrate concentration the velocity of the reaction is directly proportional to the substrate concentration. This is first order reaction
Zero order reactions
At high concentration the velocity of the reaction is independent of substrate concentration. This is zero order reaction
Substrate concentration on the reaction rate :
3. Effect of temperature
The velocity of enzyme reaction increases when the temperature is increased reaches a maximum & then falls
The temperature at which maximum amount of substrate is converted to the product per unit time is called the optimum temperature
Effect of temperature
Increase in velocity of the reaction
Increase in temperature results in high activation energy of the molecules & more molecular collision & interaction for the reaction to proceed faster
Decrease in velocity of the reaction
When the temperature is increased more than the optimum temperature Denaturation of the enzyme occurs
4. Effect of pH
The pH decides the charge on the amino acid residues at the active site. The net charge on the enzyme would influence the substrate binding & catalytic activity
The accumulation of reaction products generally decreases the enzyme velocity.
The products combine with the active site of the enzyme & form a loose complex & thus inhibit the enzyme activity.
5. Effect of product concentration
Activators: Substances that increases the enzyme activity.
Eg : Zinc activates carbonic anhydrase NAD+ activates LDH
Inhibitors: substances that decreases the enzyme activity
Eg : Fluoride inhibits Enolase.
Cyanide inhibits cytochrome oxidase.
6.Factors affecting enzyme activity
Specificity of enzyme activity
Sterospecificity Reaction specificity Substarte specificity – Absolute Relative Broad
The same substrate can undergo different type of reaction. Each reaction is catalysed by a separate enzyme
Pyruvate Acetyl coAPDH
LactateLDH
Oxalo acetate
Carboxylas
eTransaminase
Alanine
Malate
Mal
ic e
nzym
e
Reaction specificity
Substrate specificity
Absolute specificity : Enzymes that can act only on one substrate & can catalyse only one reaction
Eg : Glucose Glucose-6-phosphate
Group specificity : Carboxy peptidase & amino peptidase are exopeptidase which hydrolyse peptide bond in the vicinity of free COOH or NH2 groups respectively
Broad specificity : Hexokinase acts on Glucose, Fructose, Mannose etc
Glucokinase
Michaelis –menten graph
X- axis – substrate concentration
Y-axis – velocity of the reaction (V)
Vmax = maximum velocity a reaction can reach. Directly proportional to amount of the enzyme and substrate
Km = substrate conc at the half maximal velocity.
Enzyme concentration is kept constant. As the substrate concentration increases the velocity increases but after some conc.. The graph flattens due to enzyme saturation. The highest point on the graph is called Vmax.
Representation of enzymes at various substrate concentration
Michaelis menten equation
Vi =Vmax [S]
Km + [S]
1) When substrate conc < Km value (point A), Vi will be directly proportional to substrate conc
2) When substrate conc = Km value(point B), vi wil be half the maximal velocity
3) When substrate conc > Km value(point C), Vi wil be equal to Vmax and independent of substarte conc on further increases.
What does the Km value say about a reaction?
High Km value means – the enzyme has low affinity for the substrate .
We have to load a large amount of substrate for the reaction to attain Velocity.
Low Km value means - the enzyme has very high affinity for substrate.
Even if small amounts of substrates are there the reaction will attain velocity
Affinity / binding is inversely proportional to Km value.
Binding is good when shapes of both enzyme and substrate match
Hill’s equation;
Y=ax+b
Conversion of michaelis mentan equation which is hyperbolic to hill’s equation which is linear.
Lineweaver –Burk Double reciprocal plot
X axis = 1/S
Point of intersection on x axis = (-1/Km) value
Y axis = 1/V
Point of intersection on Y-axis(slope) = 1/Vmax
Inverse plot of michaelis menten – both S conc and velocity plotted by taking 1/S and 1/V on x and y axis. This was done to make the equation linear.
Enzyme inhibition
A substance that can inhibit enzyme activity is called enzyme inhibitor:
Types : 1. Competitive inhibition2. Non-competitive inhibition3. Uncompetitive inhibition4. Mixed inhibition
Competitive inhibition
So in competitive inhibition:
Km is increased Vmax remains
unaltered because the number of enzymes remain the same.
Inhibitor resembles substrate in shape.
Substrate competes with catalytic site.
Inhibition is reversible because the inhibitor forms non-covalent bonds
Inhibition can be reversed by increasing substrate conc..
Competitive inhibition:
Lineweaver plot:
Michaelis menten plot :
Methanol Poisoning
“Wood” alcohol and antifreeze contain high methanol concentrations
Methanol poisoning causes decreased blood pressure and body temperature, and an increase in respiratory rate
Methanol + NAD+ Formaldehyde + NADH + H+
Ethanol + NAD+ Acetaldehyde + NADH + H+
Alcohol dehydrogenase has a slightly lower Km for ethanol compared to methanol
The additional benefit of the ethanol reaction is that acetaldehyde is less physiologically damaging than formaldehyde, which is toxic to the retina and can cause permanent blindness if doses are high for prolonged periods of time
Displaced methanol can be safely excreted by the kidneys
Alcohol Dehydrogenase
Alcohol Dehydrogenase
Methotrexate Structural analogue of folate inhibits folate reductase
Dicoumarol Structurally similar to Vit Kanticoagulant
Sulfonamide antibiotics structural analogues of PABA inhibits folate synthesis in bacteria Non-toxic to humans humans don’t synthesize PABA
Non-competitive inhibition:
Inhibitors bind to site other than substrate binding site.
Covalent bond formation – occurs
Loss of enzyme activity.
So Vmax decrease but Km unaltered.
Non-competitive inhibition
Michaelis menten plot: Lineweaver plot:
Uncompetitive inhibition
Inhibitor binds to the enzyme substrate complex.
Increased Substrate affinity apparently decreases the Km but Vmax is decreased because the enzyme will take a long time to separate from the complex.
Lineweaver plot:
Km value decreases
Vmax also decreases
Uncompetitive inhibition
Summary of inhibition:
Enzyme inhibition Km Vmax
Competitive inhibition
Increases Unaltered
Non competitive inhibition
Unaltered Decreases
Uncompetitive inhibition
Decreases Decreases
Mixed Increases Decreases
Enzyme regulation
The process by which cells can turn on, turn off, modulate the activities of various metabolic pathways
For coordinating various processes as per the physiological needs of the body.
Every pathway has enzymes which are the rate limiting/ or key enzymes
Types of enzyme regulation
Coarse regulation – Regulating enzyme activity
Fine regulation – Regulating enzyme concentration.
Coarse Regulation:
Covalent modification
Allosteric regulation
Feed back inhibition
Compartmentalization
Fine Regulation:InductionRepression
Types of enzyme regulation
Allosteric Regulation
These are the enzymes having one “Catalytic site” & another separate site called “Allosteric site” where the modifier binds & regulate the enzyme activity.
Catalytic site (substrate )
Allosteric site (allosteric compound)
Enzyme
Substrate
Allosteric activator
Enzyme Product
+
Enzyme substrate
Complex
Allosteric Activation
Enzyme
Substrate
Allosteric inhibitor
No enzyme substrate
Complex
Allosteric inhibition
Sigmoid curve
Positive modifier
Substrate concentration
Negative modifier
Allosteric regulation
• Addition of a group to the enzyme by a covalent bond or removal of a group by cleaving a covalent bond
Types
Covalent modification
Adenylation - deadenylation
Ribosylation
Methylation
Acetylation
Phosphorylation-dephosphorylation
Feedback inhibition
The process of inhibiting the first step by the final product, in a series of enzyme catalysed reactions of a metabolic pathway
A B C D Ee1 e2 e3 e4
E (-)
Compartmentalization
Localisation of enzyme
Metabolic pathway
Cytoplasm Glycolysis, fatty acid synthesis
Mitochondria Krebs cycle, ETC, Fatty acid oxidation
Peroxisomes Long chain fatty acid oxidation
Both mitochondria & cytosol
Urea cycle, Heme biosynthesis