Chapter 5: Enzymes and enzyme kinetics

Learning objectives:
• Describe the characteristics and major types
of enzymes

• Understand how enzymes regulate

biochemical reactions in our body

• Appreciate enzyme kinetics as an approach to

understand the mechanism of enzyme-
catalyzed reactions

• Understand the relationship between enzyme

reaction rate and substrate concentration in
terms of Michaelis-Menten equation

• Understand the major factors affecting

enzymatic reactions
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http://karimedalla.wordpress.com/2012/10/16/3-6-7-6-enzymes/
 What are enzymes?

• Enzymes = Biological catalysts which can

speed up the rate of biochemical reactions

Enzyme
• Substrate Product

• Catalytic activity depends on the integrity of

the active site

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http://students.cis.uab.edu/clight/finalprojectwhatisanenzyme.html
Characteristics and major types
of enzymes

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 Characteristics of enzymes
1. Highly specific: Catalyze a single reaction

2. Speed up reaction in either direction (e.g. carbonic

anhydrase)
Forward (Tissue)
CO2 + H2O H2CO3
Backward (Lungs)

3. Protein in nature; can be denatured at high

temperatures or extreme pH values  Disruption of
active site and loss of function

4. Remain unchanged after reaction  Small amount is

enough

5. May require the presence of other substances in order

to function (e.g. cofactors) 4
http://chubbylemonscience.tumblr.com/page/13
 Enzyme cofactors and holoenzyme

• Cofactors are classified into 2 types:

1. Inorganic ions (e.g. Fe2+, Mg2+, or Zn2+)
2. Organic molecules = Coenzymes

• Most cofactors bind loosely to the protein

portion (i.e. apoenzyme) of enzymes

• Holoenzyme (Completely active enzyme)

= Apoenzyme (enzyme protein) + cofactors

http://www2.raritanval.edu/departments/Science/full-
time/Weber/Microbiology%20Majors/Chpater5/chapte 5
r5sub/chapter5sub_print.html
Common enzyme cofactors: Inorganic ions
(for reference)

Cofactor (ions) Enzyme

Cu2+ Cytochrome oxidase
Fe2+/Fe3+ Catalase, peroxidase
Ni2+ Urease
K+ Pyruvate kinase
Zn2+ Alcohol dehydrogenase

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 Common enzyme cofactors: Organic molecules
(for reference)

• Act as transient carriers of specific functional groups

• Mostly derived from vitamins or organic nutrients obtained in small

amounts from our diet

Coenzyme Examples of Dietary precursor

chemical groups
transferred
ATP Phosphate group Glucose
Coenzyme A Acyl groups Vitamin B5
Flavin adenine dinucleotide (FAD) Electrons Vitamin B2
Pyridoxal phosphate Amino groups Vitamin B6

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 Classification of enzymes (for reference)
• Named by adding the suffix “-ase” to the name of their substrate
(e.g. urease catalyzes the breakdown of urea)

• Six major classes:

Enzyme class number Type of reaction catalyzed

1. Oxidoreductases Oxidation/reduction (e.g. dehydrogenases)

2. Transferases Transfer chemical groups (e.g. kinases)
3. Hydrolases Hydrolysis (e.g. sucrase)
4. Lyases Addition of groups to double bond or formation of
double bonds by removal of groups (e.g. synthases)
5. Isomerases Rearrangement of groups within a molecule to form
isomers (e.g. glucose 6-phosphate isomerase)
6. Ligases Ligation requiring ATP (e.g. DNA ligase)

• Most enzymes catalyze the transfer of electrons, atoms, or functional

groups
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http://www.fastbleep.com/biology-notes/40/116/1159
 How do enzymes work?
• General equation: E + S ES EP E+P
ES = enzyme-substrate complex; EP =
enzyme-product complex

• Enzymes speed up biochemical reactions by

lowering the activation energy (i.e. energy
barrier)

• Enzymes do not change ∆G (i.e. free energy

difference between the products and
reactants)

• -ve ∆G = energy-releasing (or spontaneous)

reaction
 Favors product formation because
reaction moves to lower energy state http://www.rci.rutgers.edu/~uzwiak/GBSummer12/GB101Summer1
2Lect/GB101Summer_Lect11.htm
 Products are more stable 9
http://blog.dearbornschools.org/renkomapbio/files/2010/10/enzyme-curve-2.jpg
 Lock and key hypothesis

• Specificity of the enzyme (lock) and its substrate (key) comes

from their complementary shapes

• Substrate fits exactly with the active site of the enzyme during
the complex formation

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http://lc.brooklyn.cuny.edu/smarttutor/corc1321/energy.html
 Induced-fit model
• Enzyme and substrate do not have perfect complementary
structure

• Active site forms a complementary shape to the substrate only

after substrate binding  Induced fit

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http://lc.brooklyn.cuny.edu/smarttutor/corc1321/energy.html
Effect of substrate concentration
on enzyme kinetics:
Michaelis-Menten equation

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 Substrate Concentration

• Enzyme kinetics = Study on how rate of

the enzymatic reaction changes according
to experimental parameters (e.g. [S])

• At low [S], initial rate (V0) increases

linearly with increasing [S]

• At high [S], increase in V0 slows down and

eventually reaches a plateau
 Maximum velocity (Vmax)

13 5/e,
Nelson and Cox, Lehninger principles of biochemistry,
New York : W.H. Freeman, c2008.
 Relationship between substrate concentration and
reaction rate: Michaelis-Menten equation

• Michaelis-Menten equation:
V0: Initial velocity
Vmax: Maximum velocity
[S]: Substrate concentration
Km: Michaelis-Menten constant

• Considering when V0 is half of Vmax:

• Dividing Vmax on both sides:

• Solving for Km:

• Km = [S] at which V0 is half of Vmax

14 5/e,
Nelson and Cox, Lehninger principles of biochemistry,
New York : W.H. Freeman, c2008.
 Implications of Michaelis-Menten equation

• Michaelis-Menten equation:

• At low [S]: Km >> [S]; Km + [S] ≈ Km,

 V0 increases linearly with [S] (slope = Vmax/Km)

 Active sites are available for substrate binding

• At high [S]: [S] >> Km; Km + [S] ≈ [S],

 Vmax is observed at high [S]

 All active sites are used up by substrate molecules
(i.e. Enzyme is the limiting factor)

15 5/e,
Nelson and Cox, Lehninger principles of biochemistry,
New York : W.H. Freeman, c2008.
 Transformation of Michaelis-Menten equation:
Double-reciprocal plot (Lineweaver-Burk equation)

• Michaelis-Menten equation:

• Taking reciprocal of both sides:

• Separating components of the numerator on the right side:

• Simplifying: (Lineweaver-Burk equation)

16
Nelson and Cox, Lehninger principles of biochemistry, 5/e, New York : W.H. Freeman, c2008.
 Why double-reciprocal plot is useful?

• A plot of V0 versus [S] yields a hyperbolic curve

 Vmax is achieved at infinite [S]
 Impossible to accurately determine Vmax

• By measuring V0 at different substrate concentrations

and then plot 1/V0 against 1/[S]

• A plot of 1/V0 versus 1/[S] will give a straight line (in

the form of y = mx + c; m = slope; c = Y-intercept):
Slope = Km/Vmax
X-intercept = -1/Km
Y-intercept = 1/Vmax
 A more accurate Vmax value can be obtained
17
Nelson and Cox, Lehninger principles of biochemistry, 5/e, New York : W.H. Freeman, c2008.
Other factors which affect the
rate of enzymatic reactions

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 Enzyme concentration

• Enzymatic rate is directly proportional to the enzyme concentration

under suitable conditions, provided that an excess of free substrate
molecules is present

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http://www.worthington-biochem.com/introbiochem/enzymeconc.html
 pH

• Enzymes have optimal activity at

specific pH (2- 10)

• pH lower or higher than the

optimal pH
 Enzyme activity decreases

• Pepsin: Secreted by stomach

(optimal pH = 2)

• Trypsin: Produced in pancreas;

functions well in alkaline condition
in duodenum (optimal pH = 8)

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http://www.uic.edu/classes/bios/bios100/lecturesf04am/lect04.htm
 Temperature

• Q10 = reaction velocity at (T+10)°C ≈ 2

reaction velocity at T°C
 A rise of 10°C in temperature doubles the
reaction rate (between 5-40°C)

• ↑ temperature  ↑ K.E. of substrate

 More collisions of the substrate with the
active site
 ↑ formation of ES complex and product

• In humans, most enzymes have optimal

temperature at _________

• Higher than optimal  ↓ rate due to

alteration of active site (______________)
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http://www.bbc.co.uk/schools/gcsebitesize/science/add_ocr_
pre_2011/homeostasis/importancerev4.shtml
 Enzyme inhibitors

• Reduce enzymatic reaction rate

• Two types of inhibitors: reversible and

irreversible

• 1. Reversible inhibitors
- Effect is temporary
- No permanent change to enzyme
- Removal of these inhibitors can restore
enzymatic activity
- Further divided into 2 types:
(a) Competitive
(b) Non-competitive

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http://www.tutorialz360.com/wp-content/uploads/2012/11/inhibitors.jpg
 Enzyme inhibitors
• Competitive inhibitors
- Compete with the substrate for the enzyme active site
 Km↑
- Effect of the inhibitor decreases when [S] increases
 Vmax unchanged

• Non-competitive inhibitors
- Bind to an allosteric site (distinct from the active site)
 No competition between the substrate and inhibitor for
enzyme binding
- The substrate can still bind to enzyme (Km unchanged),
but the enzyme cannot catalyze the reaction (Vmax ↓)

23
https://bio.libretexts.org/Bookshelves/Biochemistry/Book%3A_Biochemistry_Free_and_Easy_(Ahern_and_Rajagopal)/04%3A_Catalysis/4.10%3A_Enzyme_Inhibition
 Enzyme inhibitors

• 2. Irreversible inhibitors
- Destroy a functional group on an
enzyme (e.g. DIFP destroys -OH
group of Ser at position 195 of
chymotrypsin)

- Formation of a covalent bond

between enzyme and inhibitor
 Destroy active site
 Permanent loss of catalytic
activity

24 5/e,
Nelson and Cox, Lehninger principles of biochemistry,
New York : W.H. Freeman, c2008.
 Kinetic tests for enzyme inhibition mechanism

• A double-reciprocal plot can

determine the enzyme
inhibition mechanism

• Slope of the line = Km/Vmax

• Competitive inhibition:
Vmax unchanged; Km ↑
 ↑ slope; Y-intercept
unchanged

• Non-competitive inhibition:
Km unchanged; Vmax ↓
 ↑ slope; X-intercept
unchanged
25
http://themedicalbiochemistrypage.org/enzyme-kinetics.php#inhibitors
 Case study: Test your understanding of enzyme kinetics

Q1: Hexokinase catalyzes the phosphorylation of glucose and fructose by ATP.

Km for glucose = 0.13 mmol L−1
Km for fructose = 1.3 mmol L−1
Vmax is the same for both glucose and fructose and that the enzyme displays
Michaelis-Menten kinetics.

(a) Calculate the normalized initial velocity of the reaction (i.e. V0/Vmax) for each
substrate when [S]0 = 0.13 mmol L−1.

(b) For which substrate does hexokinase have the greater affinity?

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