Enzyme Catalysis 1 Enzyme Catalysis 2

Introduction Contents
Catalysis in biochemical reactions 0. General concepts
Essential role in living organisms 1. Enzyme action
Important role in science and technology 2. Kinetics of enzyme catalytic reactions
3. Inhibition of enzyme actions
4. The effect of pH
5. Enzyme activation by metal ions
6. Regulatory enzyme
J. E. House: Principles of Chemical Kinetics, Academic Press 2nd Ed. , 2007, Chapter 6

0. Terminology 3 0. Enzymes 4

Proteins = polypeptides = polymers contn. amino acids

Reactants named substrates
Catalysts named (generally) enzymes
(specifically) apoenzymes (alone)
Supporting species named co-factors (coenzymes,
prosthetic groups, and metal ions)
(Catalysts + supporting species) named holoenzymes
Opposing species named inhibitors
 0. Enzymes 5 6

Amino acids

1. Lock and key model 7 1. Lock and key model 8

 1. Enzyme action 9 1. Enzyme action 10

Specificity (selectivity) – 4 main types

Substrate Enzyme Product
Absolute specificity: Just one reaction catalyzed Hydrogen peroxide Catalase Oxygen and water
Group specificity: Just a single type of functional group Starch Amylase Maltose
(in substrates) catalyzed
Maltose Maltase Glucose
Linkage specificity: Just a single type of bond (in
substrates) catalyzed Protein Pepsin Peptides

Stereochemical specificity: Just stereoisomer (of Peptides Protease Amino acids

substrates) catalyzed Fats Lipase Fatty acids & glycerol

1. Characteristics 11 1. Characteristics 12

Enzymes are protein materials:

Temperature sensitive: upper limit ca. 40 oC – protein

denaturation → catalytic activity loss
Optimum catalytic activity in narrow temp. ranges
pH specificity: “Free” (original) might become
protonated (low pH) or de-protonated (high pH)
Optimum catalytic activity in narrow pH ranges
Dependence of most enzyme-catalyzed Dependence of most enzyme-catalyzed
reactions on temperature reactions on pH
 2. Kinetics of enzyme catalytic reactions 13 2. Kinetics of enzyme catalytic reactions 14
2 main steps in enzyme catalytic processes:

In general similar description as another catalytic reaction types

2. Michaelis–Menten analysis 15 2. Michaelis–Menten analysis 16

In most cases, at least in the early stages, the concentration of the

Et: Total/Initial enzyme concentration (at t = 0)
product is low so that the rate of the reverse reaction characterized by
E: Enzyme concentration at t
the rate constant k-2 can be neglected.
Mass balance: E = [E]t – [ES]
𝑑[𝑃]
𝑅= = 𝑘! [𝐸𝑆]
𝑑𝑡 𝑑[ES]
= 𝑘" ( 𝐸 $ − 𝐸𝑆 ) 𝑆 − (𝑘#" + k ! )[𝐸𝑆] = 0
𝑑𝑡
𝑑[ES]
= 𝑘" 𝐸 𝑆 − (𝑘#" + k ! )[𝐸𝑆] = 0
𝑑𝑡
𝑘" E [S] 𝑘" 𝐸 $ [S]
[ES] = [ES] =
(𝑘#" + k ! ) (𝑘" [𝑆] + 𝑘#" + k ! )
Already known in method supposing constant intermediate concentration
 2. Michaelis–Menten equation 17 2. Michaelis–Menten equation 18

𝑘" 𝐸 $ [𝑆] 𝐸$
[ES] = Rmax [S] k−1 + k2
[𝐸𝑆] = 𝑘 + k! R= Km =
(𝑘" [𝑆] + 𝑘#" + 𝑘! ) (1 + #" ) [S] + K m k1
𝑘" [𝑆]

k−1 + k2 [E]t [E]t [S] 1/ K m << [S] R → Rmax = k2 [E]t

[ES] = =
Define: Km = K [S] + K m
k1 1+ m All of the enzyme bound to the substrate, reaction of the zero-order kinetics
[S]
The Michaelis constant
Rmax [S]
2 / K m >> [S] R≈ Reaction of the 1st order kinetics
Define: Km
d[P] [E]t [S] Rmax [S]
R= = k2 [ES] = k2 Rmax = k2 [E]t R=
dt [S] + K m [S] + K m Km depends on enzyme, substrate, reaction conditions (pH,
temperature, etc.)

2. Michaelis–Menten equation 19 2. Lineweaver–Burk analysis 20

1/R = y
3 / K m = [S] R [S]
R = max
[S] + K m

Rmax Slope = Km/Rmax

R = 1/Rmax
2
1 1 K 1
Km = the substrate conc. corresponding to the half of the maximal reaction rate = + m × 1/[S] = x
R Rmax Rmax [S]
The substrate conc. needed to occupy half of
the available (catalytic) sites on the enzyme 1 1 Km
The Michaelis constant
The relative binding affinity substrate –
= +
R k2 [E]t k2 [E]t [S]
(catalytic) sites on the enzyme
 2. Hanes-Wolf plot 21 2. Eadie–Hofstee analysis 22

Rmax [S] Rmax [S]

R= R=
[S] + K m [S] + K m

RK m + R[S] = Rmax [S]

[S] Km [S] Km [S]

= + = +
R Rmax Rmax k2 [E]t k2 [E]t R
R = Rmax − K m
[S]

3. Inhibition of enzyme actions 23 3. Competitive inhibition 24

Partial or even total loss of activity of enzyme The inhibitor & substrate compete with each other to bind on
the active sites of the enzyme in question.

1/ Competitive inhibition
2/ Noncompetitive inhibition
3/ Uncompetitive inhibition
 3. Competitive inhibition 25 3. Competitive inhibition 26

Ki = 1/K: the equilibrium constant for dissociation of the EI complex

3. Competitive inhibition 27 3. Non-competitive inhibition 28

 3. Non-competitive inhibition 29 3. Uncompetitive inhibition 30

The inhibitor does not bind on the active sites, but its bonding on The inhibitor binds on the enzyme-substrate complexes and
another site affects the enzyme conformation so the originally active stabilizes them so they could not break down to products.
sites become inactive.

A Lineweaver–Burk plot

A Lineweaver–Burk plot

4. The effect of pH 31 4. The effect of pH 32

H+ acts as a competitive inhibitor

Enzyme Source Optimum pH
u pepsin gastric mucosa 1.5
u sucrase intestine 6.2
u catalase liver 7.3
u arginase beef liver 9.0
K2, K1, Ks are the equilibrium constants for dissociation of the EH,
u alkaline bone 9.5
EH2+ and EHS complex, respectively.
phosphatase
 4. The effect of pH 33 4. The effect of pH 34

Total enzyme concentration from the mass balance Formal Michaelis-Menten equation

Ø Each enzyme has an

optimum pH range.

A plot of -lgKm as Ks = 5.0 x10-4, K1 = 10-5 mol/L & K2 = 10-7 mol/L

5. Enzyme activation by metal ions 35 6. Regulatory enzyme 36

Enzymes involved in metabolic pathways

Reaction rates do not follow the Michaelis-Menten kinetics
Plots reaction rate vs. [substrate] of sigmoidal shape

The actual catalyst is the enzyme-metal complex Nonregulatory enzymes

Rs = 81
(Michaelis-Menten kinetics)
KEM = km/k-m Positive cooperativity –
Substrate at 0.9 R 012
M: Metal ion 𝑅" = Rs < 81 reaction rates increase with
Substrate at 0.1 R 012
substrate conc. more

Negative cooperativity –
Rs > 81 reaction rates increase with
substrate conc. less
 6. Regulatory enzyme 37 Case study- Sulfa Drugs 38

The Hill equation K’ = binding constant

n = number of occupied sites on the enzymes • Illnesses caused by invading microorganisms like bacterium can be
n = 1 noncooperativity
combated using a competitive inhibitor called an antimetabolite.
n < 1 negative cooperativity
n > 1 positive cooperativity

• Folic acid is a coenzyme in many biosynthetic processes like

synthesis of amino acids and nucleotides.

When R = Rmax/2 →

Case study- Sulfa Drugs 39 Case study- Sulfa Drugs 40

O
Folic acid obtained from C
OH Ø In 1930 it was discovered that sulfanilamide, along with
1/ the diet or
H N sulfapyridine and sulfathiazole, could kill many types of harmful
p-aminobenzoic acid
2/ microorganisms in the intestinal tract H bacteria and help cure several diseases.

Ø The bacteria are tricked into using the sulfa drugs instead of
Microorganisms make folic acid from PABA.
PABA.
H2N N N
O O
Ø They make a molecule that also has a folic acid type of structure
N NH C NH CH C OH
N
CH2 but is not exactly the same.
OH
CH2
C OH
O
 41 Case study- Sulfa Drugs 42

• When they try to use this fake folic acid as a coenzyme,

not only doesn’t it work, it is now a competitive
inhibitor.

• Many of the bacteria’s amino acids and nucleotides cannot

be made, and the bacteria die.

Penicillin: War of Enzyme against Enzyme 43 Penicillin: War of Enzyme against Enzyme 44

v Produced by mold, it prevents growth of bacteria by v Penicillin is an example of a beta-lactam.

successfully competing for active sites on an enzyme that v It has within its structure the beta-lactam ring.
bacteria need for cell wall production.
v Over the past 15-20 years a new strain of bacteria has
1. Bacteria need the enzyme transpeptidase to make their cell developed that can resist penicillin. They contain an enzyme
walls rigid and cross-linked. (penicillinase) that opens the beta-lactam ring and renders the
2. Penicillin takes control of transpeptidase. penicillin ineffective.
3. Bacteria cell walls are not cross-linked and the contents of
the bacteria cells cannot be held in.
4. Cytoplasm spills out, and the bacteria die.
Penicillin: War of Enzyme against Enzyme 45

By changing the R group, science has found a

way to prevent this from happening.