Enzymes are globular proteins that act as biological catalysts, speeding up chemical reactions without being consumed. They are typically named after their substrate with the suffix "-ase". Enzyme activity can be monitored by measuring changes in substrate or product concentration. Mass spectrometry provides an alternative detection method without needing a chromophore. The enzyme binds its substrate at the active site, forming an enzyme-substrate complex. This lowers the activation energy and allows the reaction to proceed, with the unaltered enzyme then dissociating to catalyze more reactions. Kinetic analysis reveals the individual reaction steps and how enzyme activity is controlled.

1. Module 5
Enzyme

2. Enzyme
• An enzyme is a globular protein which acts as a
biological catalyst by speeding up the rate of a
chemical reaction
• Enzymes are not changed or consumed by the
reactions they catalyse and thus can be reused
• Enzymes are typically named after the molecules
they react with (called the substrate) and end
with the suffix ‘-ase’
• For example, lipids are broken down by the
enzyme lipase

3. How to monitor enzyme catalyzed
reactions
• Enzyme catalysis is detected by measuring either the
appearance of product or disappearance of
reactants.
• Enzyme assays are tests developed to measure
enzyme activity by measuring the change in
concentration of a detectable substance.
• Mass spectrometry is a rapid, sensitive, and accurate
quantitative approach for the direct monitoring of
enzyme-catalyzed reactions that does not require a
chromophore or radiolabeling and thus provides a
viable alternative to existing analytical techniques.

4. How does an enzyme catalyze reactions
Enzyme reactions typically occur in aqueous solutions (e.g.
cytoplasm, interstitial fluid, etc.)
• Consequently, the substrate and enzyme are usually moving
randomly within the solution (Brownian motion)
• Sometimes an enzyme may be fixed in position (e.g.
membrane-bound) – this serves to localise reactions to
particular sites
Enzyme Catalysis
Enzyme catalysis requires that the substrate be brought into
close physical proximity with the active site
• When a substrate binds to the enzyme’s active site, an
enzyme-substrate complex is formed
• The enzyme catalyses the conversion of the substrate into
product, creating an enzyme-product complex
• The enzyme and product then dissociate – as the enzyme was
not consumed, it can continue to catalyse further reactions

6. Enzyme classification

7. Mechanism of enzyme action
The catalytic efficiency of enzymes is explained
by two perspectives:
• Thermodynamic changes
• Processes at the active site

8. Thermodynamic Changes
• All chemical reaction have energy barrier
between reactant and products.
• The difference in transitional state and
substrate is called activational barrier.

9. Thermodynamic Changes

10. Thermodynamic changes overview

11. Processes at the active site

12. Covalent catalysis
• Enzymes form covalent linkages with substrate
forming transient enzyme-substrate complex with
very low activation energy.
• Enzyme is released unaltered after completion of
reaction.

13. Acid base catalysis
• Mostly undertaken by oxido- reductases
enzyme.
• Mostly at the active site, histdine is present
which act as both proton donor and proton
acceptor.

14. Catalysis by proximity

15. Catalysis by bond strain
• Mostly undertaken by lyases.
• The enzyme-substrate biding causes
reorientation of the structure of site due to in
a strain condition.
• Thus transition state is required and here
bond is unstable and eventually broken.
• In this way bond between substrate is broken
and converted into products.

16. Enzyme kinetics
• It is a branch of biochemistry in which we study the
rate of enzyme catalyzed reactions.
• Kinetics analysis reveals the numbers and order of
the individual steps by which enzymes transform
substrate into products.
• Studying an enzymes kinetics in this way can reveal
the catalytic mechanism of that enzyme, its role in
metabolism, how its activity is controlled, and how a
drug or an antagonist might inhibit the enzyme.

17. Enzyme kinetics
When an enzyme is added to a substrate, the
reaction that follows occurs in three stages with
distinct kinetics:

18. Enzyme kinetics

19. Enzyme kinetics
The pre-steady state phase is very short, as
equilibrium is reached within microseconds.
Therefore, if you measure the rate in the first
few seconds of a reaction, you will be
measuring the reaction rate in the steady
state. This is the rate used in Michaelis-
Menten Kinetics.

20. Kinetic parameters
• The 'Kinetic parameters' subsection is mostly
used to describe kinetic data, such as
Michaelis-Menten constant (KM) and
maximal velocity (Vmax).

21. Importance of chemical kinetic
• It helps to explain how enzymes work
• It helps to predict how enzymes behave in
living organisms.
• To understand and predict the metabolism of
all living things.
• Through this we know the rate of a reaction.
• Kinetic parameters are used to compare
enzyme activities.

22. RNA Catalysis
• The best-characterized ribozymes are the self-splicing
group I introns, RNase P, and the hammerhead
ribozyme.
• Most of the activities of these ribozymes are based on
two fundamental reactions: transesterification and
phosphodiester bond hydrolysis (cleavage).
• The substrate for ribozymes is often an RNA molecule,
and it may even be part of the ribozyme itself. When its
substrate is RNA, an RNA catalyst can make use of
base-pairing interactions to align the substrate for the
reaction.

23. • Ribozymes vary greatly in size. A self-splicing group I
intron may have more than 400 nucleotides. The
hammerhead ribozyme consists of two RNA strands
with only 41 nucleotides.
• As with protein enzymes, the three-dimensional
structure of ribozymes is important for function.
• Ribozymes are inactivated by heating above their
melting temperature or by addition of denaturing
agents or complementary oligonucleotides, which
disrupt normal base-pairing patterns.
• Ribozymes can also be inactivated if essential
nucleotides are changed.