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Lecture

Chemical and Biochemical Engineering 2290A/B Lecture Notes - Molar Attenuation Coefficient, Competitive Inhibition, Enzyme Kinetics


Department
Chemical and Biochemical Engineering
Course Code
CBE 2290A/B
Professor
Mita Ray

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Lab Notes - 2011
Contents
Objective ....................................................................................................................................................... 2
Theory ........................................................................................................................................................... 2
Enzymes ................................................................................................................................................ 2
Competitive Inhibition .......................................................................................................................... 4
Noncompetitive Inhibition .................................................................................................................... 4
Absorption Spectroscopy ...................................................................................................................... 6
Alkaline Phosphatase ............................................................................................................................ 8
Assay Method ....................................................................................................................................... 9
WEEK 1 .................................................................................................................................................... 10

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Lab Notes - 2011
Objective
The objective of this lab is to familiarize you with biological catalysts enzymes and their activity.
Theory
Enzymes
WHAT ARE ENZYMES?
Enzymes are protein catalysts that carry out the chemical reactions of metabolism. All chemical
reactions require activation energy to break chemical bonds and begin the reaction. The need
for activation energy acts as a barrier to the chemical reaction occurring and/or to the speed at
which it occurs.
Enzymes lower the barriers that normally prevent chemical reactions from occurring (or slow
them down) by decreasing the required activation energy. Thus, in the presence of enzymes,
reactions proceed and/or proceed at a faster rate.
Figure 1 Enzyme Activity

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Lab Notes - 2011
HOW ENZYMES LOWER ACTIVATION ENERGY
Enzymes carry out their function of lowering activation energy by temporarily combining with
the chemicals involved in the reaction. These chemicals are called the substrate.
Enzymes are specific for their substrate: A particular substrate molecule will combine
temporarily with one enzyme type, and the active site of a particular enzyme will fit only one
kind of substrate. For example, the enzyme sucrase will attach only to the substrate sucrose.
The combination is called the enzyme- substrate complex. When the enzyme and substrate
combine, the substrate is changed to a different chemical called the product. The enzyme is not
consumed or altered by the reaction.
Equation 1 Enzyme Kinetics
ENZYME KINETICS
Definition: The study of the rate at which an enzyme works is called enzyme kinetics.
The rate at which an enzyme works is influenced by several factors, e.g.,
A) Substrate concentration (the more of them available, the quicker the enzyme molecules
collide and bind with them). The concentration of substrate is designated [S] and is
expressed in units of molarity.
B) Temperature. As the temperature rises, molecular motion and hence collisions
between enzyme and substrate speed up. But as enzymes are proteins, there is an
upper limit beyond which the enzyme becomes denatured and ineffective.
C) pH. The conformation of a protein is influenced by pH and as enzyme activity is crucially
dependent on its conformation, its activity is likewise affected.
D) Presence of inhibitors.
Competitive inhibitors are molecules that bind to the same site as the substrate
preventing the substrate from binding as they do so but are not changed by the
enzyme.
Non- competitive inhibitors are molecules that bind to some other site on the enzyme
reducing its catalytic power.
E + S ES E + P
k1k2
k-1
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