3. Factors affecting enzyme activity
• The contact between the enzyme and substrate is the most
essential pre-requisite for enzyme activity
1. Enzyme concentration
2. Substrate concentration
3. Temperature
4. Hydrogen ion concentration (pH)
5. Product concentration
6. Presence of activators
7. Time
8. Light & radiation
4. Enzyme concentration
• Enzyme Concentration:
• Rate of a reaction or velocity (V) is directly proportional to the enzyme
concentration, when sufficient substrate is present.
• Velocity of reaction is increased proportionately with the concentration of
enzyme, provided substrate concentration is unlimited
5. • Substrate is a molecule on which enzyme acts.
• Velocity (Reaction rate) refers to change in the concentration of
substrate or reaction product (s) per unit time.
• It is expressed as moles/liter/sec.
• Maximum velocity (Vmax):
• It refers to maximum change in the product or substrate concentration
at a given enzyme concentration.
6. • Vmax = Kcat (e)
• e-enzyme concentration & Kcat is catalytic rate constant.
• Kcat (catalytic rate constant) – defined as the number of substrates
molecules formed by each enzyme molecule in unit time.
• Expressed as moles produced/mol enzyme/time.
8. Effect of Substrate Concentration
• Increase in the substrate concentration gradually increases the
velocity of enzyme reaction within the limited range of substrate
levels.
• A rectangular hyperbola is obtained when velocity is plotted against
the substrate concentration
• Three distinct phases of the reaction are observed in the graph (A-
linear; B-curve; C-almost unchanged.
10. Explanation
• At lower concentrations of substrate (point A in the curve), some enzyme
molecules are remaining idle.
• As substrate is increased, more and more enzyme molecules are working.
• At half-maximal velocity, 50% enzymes are attached with substrate (point B
in the curve).
• As more substrate is added, all enzyme molecules are saturated (point C).
11. • Further increase in substrate cannot make any effect in the reaction
velocity (point D).
• The maximum velocity obtained is called Vmax.
• It represents the maximum reaction rate attainable in presence of excess
substrate (at substrate saturation level).
13. Michaelis-mention Plot
• The velocity of enzyme catalysed reactions is altered as the substrate
concentration is increased.
• First order reaction:
• At low substrate concentration, velocity increases proportionally as
the concentration of the substrate is increased.
14. • Mixed order reaction:
• When the concentration of the substrate is further increased (at mid
substrate concentration), the velocity increases, but not
proportionally to substrate concentration.
• Zero order reaction:
• At high substrate concentration, the velocity is maximum & is
independent of substrate concentration.
15. Enzyme kinetics & Km value
• The enzyme (E) reacts with substrate (S) to form unstable enzyme-
substrate (ES) complex.
• The ES complex is either converted to product (P) or can dissociate
back to enzyme (E) & substrate (S).
Substrate (S) + Enzyme (E) Enzyme substrate (ES) Product (P) + Enzyme (E)
16. • K1,K2 & K3 are velocity constants.
• Km, Michaelis-mention constant is given by the formula…
E + S ES E + P
K2
K1 K3
Km =
K2 + K3
K1
17. • Michaelis-mention set up mathematical expressions for the rate of all the three
reactions in the equation.
• V as the initial rate of reaction (velocity)
• S as the initial concentration of the substrate
• V max as the maximum velocity attained with high substrate concentration when all
the enzyme molecules are occupied.
• Km as Michaelis-mention constant
V =
V max (S)
Km + (S)
18. • Measured velocity (V) is equal to ½ Vmax.
• So,
½ V max =
V max (S)
Km + (S)
Km + (S) = 2V max (S)
V max
Km + (S) = 2 (S)
Km = (S)
K stands for constant & M stands for Michaelis
19. Michaelis constant
• The formation of enzyme - substrate complex is a reversible reaction, while the
breakdown of the complex to enzyme + product is irreversible.
• 50% velocity in Y axis is extrapolated to the corresponding point on X-axis, which gives
the numerical value of Km.
• The lesser the numerical value of Km, the affinity of the enzyme for the substrate is
more.
• E.g: Km of glucokinase is 10 mmol/L and hexokinase is 0.05 mmol/L.
• 50% molecules of hexokinase are saturated even at a lower concentration of glucose.
• Hexokinase has more affinity for glucose than glucokinase.
21. Salient features of Km
• Km value is substrate concentration (expressed in moles/L) at half-maximal velocity.
• It denotes that 50% of enzyme molecules are bound with substrate molecules at
that particular substrate concentration.
• Km is independent of enzyme concentration.
• If enzyme concentration is doubled, the Vmax will be double.
• But the Km will remain exactly same.
• In other words, irrespective of enzyme concentration, 50% molecules are bound to
substrate at that particular substrate concentration.
22. • Km is the Signature of the Enzyme.
• Km value is thus a constant for an enzyme.
• It is the characteristic feature of a particular enzyme for a specific
substrate.
• The affinity of an enzyme towards its substrate is inversely related to
the dissociation constant, Kd for the ES complex.
• Km denotes the affinity of enzyme for substrate.
• The lesser the numerical value of Km, the affinity of the enzyme for the
enzyme for the substrate is more.
23. Double reciprocal plot
• Sometimes it is impractical to achieve high substrate concentrations to reach
the maximal velocity conditions.
• So, ½Vmax or Km may be difficult to determine.
• The experimental data at lower concentrations is plotted as reciprocals.
• The straight line thus obtained is extrapolated to get the reciprocal of Km.
• Called as Lineweaver–Burk Plot or Double Reciprocal Plot which can be
derived from the Michaelis-Menten equation
25. Effect of Temperature
• The velocity of enzyme reaction increases when temperature of the medium
is increased; reaches a maximum and then falls (Bell shaped curve).
• The temperature at which maximum amount of the substrate is converted to
the product per unit time is called the optimum temperature.
• Temperature is increased, more molecules get activation energy, or
molecules are at increased rate of motion.
• Their collision probabilities are increased and so the reaction velocity is
enhanced.
26. Temperature coefficient Q10
• The temperature coefficient (Q10) is the factor by which the rate of
catalysis is increased by a rise in 10°C.
• Generally, the rate of reaction of most enzymes will double by a rise in
10°C.
• When temperature is more than 50°C, heat denaturation and
consequent loss of tertiary structure of protein occurs.
• Activity of the enzyme is decreased.
27. • Most human enzymes have the optimum temperature around 37°C.
• Certain bacteria living in hot springs will have enzymes with optimum
temperature near 100°C.
29. Effect of pH
• Each enzyme has an optimum pH (usually pH between 6 and 8).
• On both sides of which the velocity will be drastically reduced.
• The graph will show a bell shaped curve
• The pH decides the charge on the amino acid residues at the active site.
• The net charge on the enzyme protein would influence substrate binding and
catalytic activity.
• Optimum pH may vary depending on the temperature, concentration of
substrate, presence of ions etc.
• Pepsin (optimum pH 1-2); ALP (optimum pH 9-10) & acid phosphatase (4-5)
31. Effect of product concentration
• The accumulation of reaction products generally decreases the enzyme
velocity.
• For certain enzymes, the products combine with the active site of enzyme
and form a loose complex and, thus, inhibit the enzyme activity.
• In the living system, this type of inhibition is generally prevented by a quick
removal of products formed
32. Effect of activators
• Some of the enzymes require certain inorganic metallic cations like Mg2+,
Mn2+, Zn2+, Ca2+, Co2+, Cu2+, Na+, K+, for their optimum activity
• Anions are also needed for enzyme activity e.g. chloride ion for amylase
• Metals function as activators of enzyme velocity through various mechanisms
combining with the substrate, formation of ES-metal complex, direct
participation in the reaction and bringing a conformational change in the
enzyme.
33. • Two categories of enzymes requiring metals for their activity
• Metal-activated enzymes
• Metalloenzyme
• Metal-activated enzymes:
• The metal is not tightly held by the enzyme and can be exchanged
easily with other ions.
• e.g. ATPase (Mg2+ and Ca2+) & Enolase (Mg2+)
34. • Metalloenzyme:
• These enzymes hold the metals rather tightly which are not readily
exchanged.
• e.g. Alcohol dehydrogenase, carbonic anhydrase, alkaline phosphatase,
carboxypeptidase and aldolase contain zinc.
• Phenol oxidase (copper)
• Pyruvate oxidase (manganese)
• Xanthine oxidase (molybdenum)
• Cytochrome oxidase (iron and copper)
35. Effect of time
• Under ideal and optimal conditions (like pH, temperature etc.), the time
required for an enzyme reaction is less.
• Variations in the time of the reaction are generally related to the
alterations in pH and temperature.
36. Effect of light and radiation
• Exposure of enzymes to ultraviolet, beta, gamma & X-rays
inactivates certain enzymes due to the formation of peroxides.
e.g. UV rays inhibit salivary amylase activity
38. Enzyme inhibitor
• Enzyme inhibitor is defined as a substance, which binds with the enzyme
and brings about a decrease in catalytic activity of that enzyme.
• They are usually specific and they work at low concentrations
• They block the enzyme but they do not usually destroy it
• Many drugs and poisons are inhibitors of enzymes in the nervous system
39. Type of Enzyme Inhibitors
Reversible
Irreversible
Type of
Inhibitors
Competitive
Uncompetitive
Non- Competitive
Active Site
Directed
Suicide / kcat
Inhibitors
40. Reversible inhibition
• The inhibitor binds non-covalently with enzyme and the enzyme inhibition
can be reversed if the inhibitor is removed.
• Binding is weak and thus, inhibition is reversible.
• Do not cause any permanent changes in the enzyme
• Subtypes:
• Competitive & Non-competitive Inhibition
41. Competitive inhibition
• The inhibitor (I) molecules resembles the real substrate (S)
• Also called as substrate analogue inhibition
• Binds to active site – forms EI complex.
• EI complex cannot rive rise to product formation.
• As long as the competitive inhibitor holds the active site, the enzyme is not available for
the substrate to bind.
• Relative concentrations of S, I determine inhibition.
E
ES
EI
E + P
No product formation
42. Binding of S & I in different Situations
• Classical Competitive Inhibition (S & I compete for the same binding
site)
Enzyme
43. • Binding of I to a distinct inhibitor site causes a conformational change in
the enzyme that distorts or masks the S binding site or vice versa.
Enzyme
I S
Enzyme
I
S
Enzyme
I
S
44. • A competitive inhibitor diminishes the rate of catalysis by reducing the
proportion of enzyme molecules bound to a substrate.
• Competitive inhibition can be relieved by increasing the substrate
concentration & maximum velocity is regained.
• A higher substrate concentration is therefore needed to achieve a
halfmaximum rate, Km increases
• High concentrations of the substrate displace the inhibitor again.
• The V max, not influenced by this type of inhibition.
45. • E. g. Malonate – structural analog of succinate-inhibits succinate
dehydrogenase.
47. • The compounds malonic acid, glutaric acid and oxalic acid, have structural
similarity with succinic acid and compete with the substrate for binding at
the active site of SDH.
• Antimetabolites:
• These chemical compounds that block the metabolic reactions by their
inhibitory action on enzymes.
• Antimetabolites are usually structural analogues of substrates and thus are
competitive inhibitors.
• They are in use for cancer therapy, gout etc.
48. Examples of competitive inhibition
Enzyme Substrate Competitive inhibitor
Succinate Dehydrogenase Succinate Malonate
Dihydrofolate Reductase 7,8-dihydrofolate Aminopterin
Xanthine Oxidase Hypoxanthine Allopurinol
Acetyl cholinesterase Acetylcholine Succinylcholine
Lactate Dehydrogenase Lactate Oxamate
HMG CoA Reductase HMG Co A HMG
52. Non-Competitive Inhibition
• The inhibitor binds at a site other than the active site on the enzyme &
causes conformational changes on enzymes or some times it may react
with functional group at the active site & inactivates the enzyme.
• This binding impairs the enzyme function.
• Inhibitor has no structural resemblance with the substrate.
• There is no competition for the active site of the enzyme molecule.
53. • There usually exists a strong affinity for the inhibitor to bind at the
second site.
• The inhibitor does not interfere with the enzyme-substrate binding.
• But the catalysis is prevented, possibly due to a distortion in the enzyme
conformation
• The inhibitor generally binds with the enzyme as well as the ES complex.
• Km value is unchanged & V max is lowered.
54. • Heavy metal ions (Ag+, Pb2+, Hg2+ etc.) can non-competitively inhibit the enzymes by
binding with cysteinyl sulfhydryl groups & inactivates the enzymes.
• Heavy metals also form covalent bonds with carboxyl groups & histidine, results in
irreversible inhibition.
• Non-competitive inhibition is also called as enzyme poisons
E + S ES
+
I
EI + S
E + P
+
I
EIS
57. Lineweaver – Burk Plot
[I]2
[I]1
No Inhibitor
1
Vmax
1
Vmaxi
1
Km
1/[s]ď‚®
1/v
• Km value is unchanged
• V max is lowered
58. Comparison between competitive & Non-competitive inhibition
Competitive Inhibition Non-competitive Inhibition
Acting on Active site May or may not
Structure of inhibitor Substrate analogue Unrelated molecule
Inhibition is Reversible Generally Irreversible
Excess Substrate Inhibition Relieved No effect
Km Increased No Change
V max No Change Decreased
Significance Drug Action Toxicological
59. Uncompetitive Inhibition
• Here inhibitor does not have any affinity for the active site of enzyme.
• Inhibitor binds only with enzyme-substrate complex; but not with free
enzyme.
• Both V max and Km are decreased
60. • UC Inhibition is rare in single-substrate reactions.
• E.g. Phenylalanine inhibits alkaline phosphatase in intestinal cells
• It is common in multi-substrate reactions
E + S E S E + P
+
I
ESI
63. • In this type, Inhibitor binds at or near the active site of the enzyme irreversibly,
usually by covalent bonds, so that it can’t subsequently dissociate from the enzyme
• The I destroys as essential functional group on the enzyme that participates in
normal S binding or catalytic action.
• As a result the enzyme is permanently inactive
• Compounds which irreversibly denature the enzyme protein or cause non-specific
inactivation of the active site are not usually regarded as irreversible inhibitors.
Irreversible inhibition
64. • These inhibitors are toxic poisonous substances.
• Iodoacetate:
• It is an irreversible inhibitor of the enzymes like papain and glyceraldehyde 3-
phosphate dehydrogenase
• Iodoacetate combines with sulfhydryl (-SH) groups at the active site of these enzymes
and makes them inactive.
• Diisopropyl fluorophosphafe (DFP) is a nerve gas developed by the Germans during
Second World War.
• DFP irreversibly binds with enzymes containing serine at the active site, e.g. serine
proteases, acetylcholine esterase.
Examples
65. Examples
• DFP (Diisopropylphosphofluoridate) is a nerve poison.
• It inactivates acetylcholinesterase that plays an important role in the
transmission of nerve impulses.
E CH2-OH + F—P=O E CH2-O- F—P=O + HF
OCH(CH3)2
OCH(CH3)2
OCH(CH3)2
OCH(CH3)2
DFP Catalytically inactive
enzyme
66. • Disulfiram (Antabuse)s a drug used in the treatment of alcoholism.
• lt irreversibly inhibits the enzyme aldehyde dehydrogenase.
• Alcohol addicts, when treated with disulfiram become sick due to the
accumulation of acetaldehyde, leading to alcohol avoidance
67. Suicidal inhibition
• This is a special type of irreversible inhibition.
• Also called as mechanism based inactivation.
• In this case, the original inhibitor (the structural analogue/competitive
inhibitor) is converted to a more effective inhibitor with the help of same
enzyme that ought to be inhibited.
• The formed inhibitor binds irreversibly with the enzyme.
• Allopurinol, an inhibitor of xanthine oxidase, gets converted to alloxanthine, a
more effective inhibitor of this enzyme.
68. • A suicide inhibitor is a relatively inert molecule that is transformed by an enzyme at
its active site into a reactive compound that irreversibly inactivates the enzyme
• They are substrate analogs designed so that via normal catalytic action of the
enzyme, a very reactive group is generated.
• The latter forms a covalent bond with a nearby functional group within the active
site of the enzyme causing irreversible inhibition.
• Such inhibitors are called suicide inhibitors because the enzyme appears to commit
suicide.
• e.g. FdUMP is a suicide inhibitor of thymidylate synthase.
Suicidal inhibition
69. • The use of certain purine and pyrimidine analogues in cancer therapy is
also explained on the basis suicide inhibition.
• 5-fluorouracil gets converted to fluorodeoxyuridylate which inhibits the
enzyme thymidylate synthase, and thus nucleotides synthesis
70. During thymidylate synthesis, N5,N10- methyleneTHF is converted to 7,8-dihydrofolate;
methyleneTHF is regenerated in two steps
71. Allosteric regulation
• The catalytic activity of certain regulatory enzymes is modified by certain low
molecular weight substances or molecules known as allosteric effectors.
• Allosteric enzyme has one catalytic site where the substrate binds and another
separate allosteric site where the modifier binds (allo = other)
• Allosteric and substrate binding sites may or may not be physically adjacent.
• The binding of the regulatory molecule can either enhance the activity of the enzyme
(allosteric activation), or inhibit the activity of the enzyme (allosteric inhibition).
72. • The binding of substrate to one of the subunits of the enzyme may enhance
substrate binding by other subunits.
• This effect is said to be positive co-operativity
• If the binding of substrate to one of the subunits decreases the activity of
substrate binding by other sites, the effect is called negative co-operativity.
• In most cases, a combination is observed, resulting in a sigmoid shaped
curve
73. The switch: Allosteric inhibition
Allosteric means “other site”
E
Active site
Allosteric
site
74. Switching off
• These enzymes have two
receptor sites
• One site fits the substrate like
other enzymes
• The other site fits an inhibitor
molecule
Inhibitor fits into
allosteric site
Substrate
cannot fit
into the
active site
Inhibitor
molecule
76. Salient Features, Allosteric Inhibition
• The inhibitor is not a substrate analogue
• It is partially reversible, when excess substrate is added.
• Km is usually increased & V max is reduced.
• The effect of allosteric modifier is maximum at or near substrate
concentration equivalent to Km.
77. • When an inhibitor binds to the allosteric site, the configuration of
catalytic site is modified such that substrate cannot bind properly.
• Most allosteric enzymes possess quaternary structure.
• They are made up of subunits, e.g. Aspartate transcarbamoylase has 6
subunits and pyruvate kinase has 4 subunits
78. Allosteric enzymes
Enzyme Allosteric Inhibitor Allosteric Activator
ALA synthase Heme
Aspartate transcarbamoylase CTP ATP
HMGCoA-reductase Cholesterol
Phosphofructokinase ATP, citrate AMP, F-2,6-P
Pyruvate carboxylase ADP AcetylCoA
Acetyl CoA carboxylase AcylCoA Citrate
Citrate synthase ATP
Carbamoyl phosphate synthetase I NAG
Carbamoyl phosphate synthetase II UTP
79. • For understanding the regulation of enzyme activity within the living cells
• To elucidate the kinetic mechanism of an enzyme catalyzing a multi-
substrate reaction
• Useful in elucidating the cellular metabolic pathways by causing
accumulation of intermediates
• Identification of the catalytic groups at the active site
• Provide information about substrate specificity of the enzyme
Importance of Enzyme Inhibition
80. Regulation of enzyme activity
• Allosteric regulation
• Activation of latent enzymes
• Compartmentation of metabolic pathways
• Control of enzyme synthesis
• Enzyme degradation
• lsoenzymes
81. Allosteric Regulation or Allosteric Inhibition
• Enzymes possess additional sites, known as allosteric sites besides the active
site.
• Such enzymes are known as allosteric enzymes.
• The allosteric sites are unique places on the enzyme molecule
• Allosteric effectors:
• The catalytic activity of certain regulatory enzymes is modified by certain
low molecular weight substances or molecules known as allosteric effectors
or modifiers bind at the allosteric site and regulate the enzyme activity.
82. • The allosteric effectors may be positive or negative effectors
• The enzyme activity is increased when a positive (+) allosteric effector
binds at the allosteric site known as activator site.
• A negative (-) allosteric effector binds at the allosteric site called inhibitor
site and inhibits the enzyme activity.
• Classes of allosteric enzyme:
• They are divided into two classes based on the influence of allosteric
effector on Km and V max
83. • K-class of allosteric enzymes:
• The allosteric inhibitor increases the Km and not the V max.
• Double reciprocal plots, similar to competitive inhibition are obtained e.g.
phosphofructokinase.
• V-class of allosteric enzymes:
• The allosteric inhibitor decreases the V max and not the Km.
• Double reciprocal plots resemble that of non-competitive inhibition e.g.
acetyl CoA carboxylase
84. Feedback regulation
• The process of inhibiting the first step by the final product, in a series of enzyme
catalysed reactions of a metabolic pathway is referred to as feedback regulation.
• The very first step (A to B) by the enzyme is the most effective for regulating the
pathway, by the final end product D.
• This type of control is often called negative feedback regulation
A B C D
86. Activation of latent enzymes
• Some enzymes are synthesized as Proenzymes or zymogens which undergo irreversible
covalent activation by the breakdown of one or more peptide bonds
• Chymotrypsinogen pepsinogen and plasminogen, are respectively- converted to the
active enzymes chymotrypsin, pepsin and plasmin.
• Certain enzymes exist in the active and inactive forms which are interconvertible
• The inter-conversion is brought about by the reversible covalent modifications, namely
phosphorylation and dephosphorylation, and oxidation and reduction of disulfide bonds
87. Examples
• There are some enzymes which are active in dephosphorylated state
and become inactive when phosphorylated e.g. glycogen synthase,
acetyl CoA carboxylase.
• A few enzymes are active only with sulfhydryl (-SH) groups
• E.g. succinate dehydrogenase, urease.
• Glutathione bring about the stability of these enzymes.
88. Compartmentation
• Generally, the synthetic (anabolic) and breakdown (catabolic) pathways
are operative in different cellular organelle.
• E.g. Enzymes for fatty acid synthesis are found in the cytosol whereas
enzymes for fatty acid oxidation are present in the mitochondria
89. Control of enzyme synthesis
• Most of the enzymes, the rate limiting ones, are present in very low concentration.
• Many rate limiting enzymes have short half-lives
• This helps in the efficient regulation of the enzyme levels.
• Constitutive enzymes (house-keeping enzymes)-The levels of which are not controlled
and remain fairly constant.
• Adaptive enzymes-Their concentrations increase or decrease as per body needs and are
well-regulated.
• The synthesis of enzymes (proteins) is regulated by the genes.
90. Induction and repression
• Induction is used to represent increased synthesis of enzyme while repression indicates its
decreased synthesis.
• Induction or repression which ultimately determines the enzyme concentration at the
gene level through the mediation of hormones or other substance.
• E.g of Induction: The hormone insulin induces the synthesis of glycogen synthetase,
glucokinase, phosphofructokinase and pyruvate kinase.
• All these enzymes are involved in the utilization of glucose.
• The hormone cortisol induces the synthesis of many enzymes e.g. pyruvate carboxylase,
tryptophan oxygenase and tyrosine aminotransferase
91. • Examples of repression:
• In many instances, substrate can repress the synthesis of enzyme.
• Pyruvate carboxylase is a key enzyme in the synthesis of glucose from
non-carbohydrate sources like pyruvate and amino acids.
• lf there is sufficient glucose available, there is no necessity for its
synthesis.
• This is achieved through repression of pyruvate carboxylase by glucose.
92. Enzyme degradation
• Every enzyme has half-life.
• It is in days while for others in hours or in minutes,
• e.g. LDH4 - 5 to 6 days;
• LDH1 - 8 to 12 hours;
• Amylase -3 to 5 hours
• The key and regulatory enzymes are most rapidly degraded.
• lf not needed, they immediately disappear and, when required, they are
93. Units of enzyme activity
• Katal:
• One kat denotes the conversion of one mole substrate per second (mol/sec).
• Activity may also be expressed as millikatals (mkat), microkatals (µkat)
• International Units (lU):
• One Sl unit or International Unit (lU) is defined as the amount of enzyme activity
that catalyses the conversion of one micromol of substrate per minute.
• Sl units and katal are interconvertible
94. Non-protein enzymes
• Ribozymes are a group of ribonucleic acids that function as biological
catalysts, and they are regarded as non-protein enzymes.
• RNA molecules are known to adapt a tertiary structure just as in the
case of proteins
• The specific conformation of RNA may be responsible for its function
as biocatalyst.
