Enzyme physiology &

kinetics
General properties & Definition
Enzymes are biological catalysts that increase the rate of
biochemical reactions.
Most enzymes are proteins
As a protein, each enzyme contains a specific amino acid sequence
(primary structure)
Resultant polypeptide chains twist (secondary structure), which
then folds (tertiary structure) and results in structural cavities
General properties & Definition
Quaternary structure refers to the spatial relationships between the
subunits in an enzyme with two or more polypeptide units.

 Quaternary structure
General properties & Definition
 Active site, often a water-free cavity, where the substrate interacts
with particular charged amino acid residues
Allosteric site—a cavity other than the active site; may bind
regulator molecules
Isoenzyme- different forms of the enzyme within the same
individual. Differ in electrophoretic mobility, solubility, or
resistance to inactivation.
General properties & Definition
Results when an enzyme is subject to posttranslational
modifications.
A non-protein molecule, called a cofactor, may be necessary for
enzyme activity
Inorganic cofactors, such as chloride or magnesium ions, are called
activators
A coenzyme is an organic cofactor, such as nicotinamide adenine
dinucleotide (NAD)
General properties & Definition
When bound tightly to the enzyme, the coenzyme is called a
prosthetic group
Holoenzyme – the enzyme portion (apoenzyme) with its respective
coenzyme
Zymogen- a proenzyme; common with digestive enzymes
General properties & Definition
Enzyme classification and
Nomenclature
Systematic name (IUB)- defines the substrate, the reaction and,
possibly, the name of any coenzyme involved
 Trivial name (recommended name ) – where former is lengthy

uricase is called urate: O2 oxidoreductase

glutamic oxaloacetic transaminase (GOT) is called L-aspartate: 2-
oxoglutarate aminotransferase.
Enzyme classification and
Nomenclature
1. Oxidoreductases. Catalyze an oxidation–reduction reaction
between two substrates
2. Transferases. Catalyze the transfer of a group other than hydrogen
from one substrate to another
3. Hydrolases. Catalyze hydrolysis of various bonds
4. Lyases. Catalyze removal of groups from substrates without
hydrolysis; the product contains double bonds
5. Isomerases. Catalyze the interconversion of geometric, optical, or
positional isomers
6. Ligases. Catalyze the joining of two substrate molecules
Class Reaction Enzymes

1. Oxidoreductases Ared + Box→Aox + Bred Dehydrogenases, peroxidases

2. Transferases A-B + C→A + B-C Hexokinase, transaminases

3. Hydrolases A-B + H2O→A-H + B-OH Alkaline phosphatase, trypsin

4. Lyases (synthases) X-A-B-Y→A = B + XY fumarase, dehydratases

Triose phosphate isomerase,

5. Isomerases A ↔ isoA
phosphogluco-mutase

6. Ligases Pyruvate carboxylase, DNA

A + B + ATP→A-B + ADP + Pi
(synthetases) ligases
Enzyme Kinetics
A chemical reaction may occur spontaneously if the free energy or
available kinetic energy is higher for the reactants than for the
products
Proceeds toward the lower energy if a sufficient number of the
reactant molecules possess enough excess energy to break their
chemical bonds and collide to form new bonds
Activation energy: the amount of energy required to break the
enzyme-substrate bond
Energy versus progression of reaction
Enzyme Kinetics
Enzyme Kinetics
The general relationship among enzyme, substrate, and product may
be represented as follows:
E + S ↔ ES* ↔ E + P
The transition state for the ES complex has a lower energy of
activation than the transition state of S alone
Enzyme Kinetics
A typical enzyme accelerates the reaction in a kinetic way that can
be given by what is called the Michaelis-Menten equation
One major influence on enzymatic reactions is substrate
concentration
The substrate readily binds to free enzyme at a low-substrate
concentration
The reaction rate steadily increases as more substrate is added, and
follows first-order kinetics*
Michaelis-Menten curve of velocity versus substrate concentration for enzymatic reaction. Km is
the substrate concentration at which the reaction velocity is half of the maximum level
Enzyme Kinetics
When product is formed, the resultant free enzyme immediately
combines with excess free substrate.
The reaction is in zero-order kinetics, and the reaction rate depends
only on enzyme concentration.
The Michaelis-Menten hypothesis of the relationship between
reaction velocity and substrate concentration can be represented
mathematically as follows:
Enzyme Kinetics
Lineweaver-Burk transformation of Michaelis Menten

which can be thought of as y = m • x + b, where 1/V is y and 1/[S]

is x
Enzyme Kinetics
Quiz 1
In a particular enzyme-catalyzed reaction, Vmax = 0.2 mol/sec and
Km = 5 mM.
Assume the enzyme shows standard Michaelis-Menten kinetics.
a) (5) What is the rate of the reaction when [S] = 10 mM?
Quiz 2

- At low concentrations of substrate, which enzyme which enzyme would be better to use ?
- At saturating concentrations of substrate, which enzyme would be better to use?
Quiz 3
The Km of the substrate is: 4, 2, 1, 0.5, 0.25
Enzyme Kinetics
Other factors that affect enzymes

 Temperature- Increasing temperature usually increases the rate of a

chemical reaction
 Each enzyme functions optimally at a particular temperature

 PH- Enzymes are proteins that carry net molecular charges

 Changes in pH may denature an enzyme or influence its ionic state
Enzyme Kinetics
Inhibitors
Competitive inhibitors - physically bind to the active site and
compete with the substrate.
The inhibition is reversible
Noncompetitive inhibitor - binds an enzyme at a place other than
the active site. May be reversible or irreversible
Uncompetitive inhibition - inhibitor binds to the ES complex. No
product
Enzyme Kinetics
Enzyme Kinetics
Enzyme Kinetics
Uncompetitive inhibition
Vmax decreased, Km appears decreased
Enzyme Regulation
Enzyme assays
Enzyme activity
Enzyme assays usually depend on the measurement of the catalytic
activity of the enzyme, rather than the concentration of the enzyme
protein itself
Enzyme activity = moles of substrate converted per unit time = rate
× reaction volume.
The SI unit is the katal, 1 katal = 1 mol s−1

A morepractical and commonly used value is enzyme unit (U) = 1

μmol min−1. 1 U corresponds to 16.67 nanokatals
Generally, three methods are followed for the estimation of various
analytes in clinical chemistry

1. End Point method or end point chemisty

2. Fixed time method or fixed time chemistry
3. Kinetic method or Kinetic Chemistry
End Point Method:
This method is employed for the estimation of analytes, which
would be completely consumed in the reaction.
In the end point methods the absorbance, at a specific wavelength,
of the complex being formed is measured
This absorbance increases against the reagent blank absorbance,
over a period of time.
This increases in absorbance continues till it reaches a stable value,
marking the end point of the reaction being reached
Fixed Type Method:
Based on the principle of the difference in absorbance between an
initial value and final value, during a specified time interval.
The time interval is optimized in such a way to minimize the
interferences with the test analytes from other analytes in the sera or
sample.
 The assumption is that a constant amount of product is produced
during the entire time period.
 Kinetic Method:
Based on the principle of measurement of the difference in
absorbance between two points over a period of specified time
during the progress of the reaction.
The assumption is that a constant amount of product is produced
during the time interval being monitored.
The delta absorbance obtained would be multiplied by an
appropriate factor for calculations, to give the quantity of the
analyte being tested in the sample.
These tests are standardized to give a linear delta absorbance over a
period of time and upto the specific linearity mentioned for each
analyte.
Kinetic Chemistries could be classified as:
a. Increasing Type: In this type, the reaction proceeds in positive
direction, where in, the initial absorbance is always lower than the
latter absorbance.

b. Decreasing Type: This type of chemistry is also called as the
negative direction type.
 Inthis method, the difference between a latter point of absorbance and an
initial point, over a specified period of time is always negative.
Why does maintaining constant temperature matter
(sometimes)?
• endpoint assays are based on using up all of the substance being
assayed
- reaction faster/slower → endpoint is same
• kinetic assays do not use up all substrate

- based on the initial rate of reaction. Higher temperature increases

rate
• higher temp → overestimate of amount/activity of substance being
assayed
Kinetic Techniques:
 Advantages:

• data is collected more quickly

• avoids interferences from slow reacting Substances

 Disadvantages

• sensitive to temperature
• measurements must be done on a time schedule
• random error tends to be more than endpoint techniques
Calculations with Kinetic Assays
Homework
END