TPN 202 BASIC FOOD CHEMISTRY AND

BIOCHEMISTRY
3 (1-2)

Coordinator:
Dr. Dian Herawati

Food Technology Study Program

Department of Food Science and Technology
IPB University

http://fst.ipb.ac.id
Internationally Accredited Study Program by IFT and IUFoST

TPN 202

Enzyme Activity

Food Technology Study Program

Department of Food Science and Technology
Course objectives

After this unit, students are expected to be able to:

• Explain the principle of enzyme activity
• Explain how to measure enzyme activity
Introduction
❖ Life depends on the
existence of powerful and
specific catalysts: the
enzymes. Almost every
biochemical reaction is
catalyzed by an enzyme.

❖ With the exception of a few

catalytic RNAs, all known
enzymes are proteins. Many
require nonprotein
coenzymes or cofactors for
their catalytic function.
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What is enzymes?

❖ A biological catalyst that promotes and speeds up a

chemical reaction without itself being altered in the
process
❖ Lower activation energy
❖ Enzyme combines with a specific substrate to a form
an enzyme-substrate complex in a lock and key (or
induced fit) concept before forming new products

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Enzyme structure
Some enzymes require no chemical
groups for activity other than their
amino acid residues

Others require an additional chemical

component called a cofactor or a
coenzyme

Some enzymes require both a

coenzyme and cofactor

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Cofactor and Coenzyme
Nomenclature of Enzymes
❖ Names usually end in –ase.
❖ Usually named after substrates they act upon
urea --- urease
lactose --- lactase
❖ or the resulting type of chemical reaction
hydrolysis --- hydrolases
oxidation --- oxidases
❖ This rule does not always apply. e.g. ficin found in
figs and papain in papayas

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Classification of Enzymes

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Basic Mechanism

Enzyme not
only recognizes
substrate, but
also induces
the formation of
transition state

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Adapted from Nelson & Cox (2004) Lehninger Principles of Biochemistry (3e) p.198
Why enzymes can lower activation energy?

It is a magic pocket

+ (1) Stabilizes transition

(2) Expels water
CoE (1) (2)
(3) Reactive groups
(4) - (4) Coenzyme helps
(3)

Juang RH (2004) BCbasics

 Contact stability

+
-

Preventing the influence of water sustains the formation of stable ionic bonds

Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.115
Additional catalytic mechanism
❖Additional catalytic mechanisms employed by enzymes
include general acid-base catalysis, covalent catalysis,
and metal ion catalysis.

❖Catalysis often involves transient covalent interactions

between the substrate and the enzyme, or group transfers
to and from the enzyme, so as to provide a new, lower-
energy reaction path.

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Active site
● Enzyme provides a catalytic surface
● This surface stabilizes transition state
● Transformed transition state to product

B
A

A B Catalytic surface

Juang RH (2004) BCbasics

 Carboxypeptidase A
(248)
(270)
Active Tyr
Glu 3 4
site
O-
ACTIVE Site for
pocket COO - H SITE specificity
+
H
H O-
C N R 5
N C C
2
Substrate O-

Juang RH (2004) BCbasics

peptide 1 + COO - +Arg (145)
chain His
(196)
Zn Glu C-terminus
(72) Check for
His (69)
C-terminal
Chymotrypsin
❖ Enzyme bind to substrate in a suitable distance and orientation with the
catalytic site
❖ Catalytic component of chymotrypsin 3 amino acids, catalytic triad, plus
oxyanion hole.
Steps contribute to transition state

Negatively charge Aspartat 102 strengthen ability of His 57's to catch H+

to gain positive charge
Steps contribute to transition state

Histidine 57 act as base by catching H+ from Ser 195

Steps contribute to transition state

Serine 195 which loose H become nucleophille and reactive. Induce

nucleophillic attack to the substrate
Steps contribute to transition state

Hidrogen bonding with H of Ser 195 changes of the position of O atom

from the trigonal planar (120o) to reach 109o. This enable transition of
tetrahedral., making oxyanion hole (backbone N-H Gly 193, Ser 195
 Steps contribute to transition state

Chemically C-O- is not stable, and possess free electron pair which return
the double bond with C. The C Atom will to degrade other bond (Oktet
Steps contribute to transition state

The protonated HisH+ now plays roles as acid. Giving H+ to substrate N, causing
breakdown of C-N, to give the product
Measuring Enzyme Activity
❖ Enzymes speed up the rate of a reaction by a definite
amount, proportional to quantity of enzyme present.
❖ Enzyme assay: the process or measuring enzyme
catalyzed reaction rate.
❖ Enzyme kinetics: mathematical analysis of how the
observed reaction rate varies with substrate
concentration; kinetic behaviour can be used to test
models of reaction mechanism (rules out wrong models)
Activity Measurement

❖ Enzyme is a catalyst, not consumed in the reaction, because the

complete reaction cycle restores the enzyme to its initial state.
❖ To measure reaction rate some property difference between
reactant and product must be identified. Rate can be determined by
measuring disappearance of reactant or accumulation of product.
Example : Trypsin

❖ Separate the products and analyze each one, but it's a lot of work.
❖ Alternatively, measure the increase in total reactive terminal amino
groups by reaction with the amino acid detection reagent,
▪ ninhydrin reacts with N-terminal amino groups, not with peptide-
bonded NH groups.
❖ The enzyme reaction creates one new terminal amino group per
peptide bond hydrolyzed
Using synthetic substrate

❖ The structure of the artificial substrate is sufficiently similar to a

peptide that trypsin can bind the lysine, and hydrolyze the amide
bond between lysine carboxylate and the the aniline NH2. The free
p-nitroaniline has a distinctive colour which is easily measured.
❖ Artificial substrates are often used for enzymes which hydrolyze
simple ester, amide or glycoside bonds.
Natural Substrate; Lactate dehydrogenase

❖ Lactate dehydrogenase oxidizes the secondary alcohol in lactate to a

carbonyl in pyruvate. This removes 2 H atoms (not H+) from the
substrate, hence the enzyme name dehydrogenase. Many metabolic
enzymes follow this naming pattern
❖ The 2 H atoms are donated to a common biological oxidizing agent,
NAD+, nicotinamide adenine dinucleotide, turning it into NADH + H+.
❖ NAD+ does not absorb ultraviolet at 340 nm
❖ NADH strongly absorbs ultraviolet at 340 nm
❖ The reaction progress can be monitored by measuring the ultraviolet
absorbance increase at 340 nm due to the formation of NADH.
Calculation
❖ Absorbance = e . c . l
❖ where e = extinction coefficient, a characteristic constant for the
absorbing substance. e = 6200 L mol-1 cm-1 for NADH at 340 nm.
c = concentration of NADH in mol / L,
l = thickness of the sample in cm (usually 1.00 cm for standard sample
cuvets).
❖ Sample calculation:
▪ A lactate dehydrogenase reaction gave an increase in absorbance of
0.31 units in one minute due to the NADH formed..
▪ Increase in [NADH] = (absorbance increase)/e . L = 0.31 / 6200 = 5.0 x
10-5 mol /L
Activity
❖ Enzyme activity = moles converted per unit time = rate x volume
❖ Enzyme activity is a function of quantity of enzyme.
❖ The SI unit is the katal,
❖ 1 katal = 1 mol s-1, but this is an excessively large unit.
❖ A more practical value is 1 enzyme unit (EU) = 1 µmol min-1 (µ = micro, x
10-6)
❖ Specific activity = moles converted per unit time per unit mass of enzyme
= enzyme activity / actual mass of enzyme present
❖ This is a measure of enzyme efficiency, usually constant for a pure
enzyme; if the specific activity of 100% pure enzyme is known, then an
impure sample will have a lower specific activity, allowing purity to be
calculated.
❖ SI: katal kg-1, Practical 1 µmol mg-1 min-1 or 1 µmol µg-1min-1.
❖ Turnover number = moles converted per unit time per mole of enzyme =
specific activity x molar mass of enzyme (with necessary unit
conversions!)
Example : Chymotripsin
At start: After 10 min:
25.0 x 10-3 mol L-1 peptide
18.6 x 10-3 mol L-1 peptide substrate,
substrate,
volume 2.5 mL,
volume 2.5 mL,
0.50 µg chymotrypsin.
0.50 µg chymotrypsin
Peptide substrate
= 6.4 x 10-3 mol L-1 in 10 minutes
consumed
Rate of reaction = 6.4 x 10-4 mol L-1 min-1
Enzyme activity = 6.4 x 10-4 mol L-1 min-1 x 2.5 x 10-3 L
(rate x volume) = 1.6 x 10-6 mol min-1
Specific activity = 1.6 x 10-6 mol min-1 / 0.50 µg
(activity / mass) = 8.0 x 10-7 mol µg-1 min-1
= 8.0 x 10-7 mol µg-1 min-1 x 25,000 x
Turnover number
106 µg mol-1
(sp. act. x molar mass)
= 2.0 x 104 min-1 = 333 s-1
Why do we need to measure enzyme activity?
The activity value (units/ml) for your enzyme is the most important
parameter when you are developing an assay. This is because the
volume (i.e. number of units) that you add will determine the amount
of substrate that is converted into product.

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Enzyme application in food industry?

Raveendran S,
Parameswaran B,
Ummalyma SB, et al.
Applications of Microbial
Enzymes in Food
Industry. Food Technol
Biotechnol.
2018;56(1):16-30.
doi:10.17113/ftb.56.01.18.
5491

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Thank you