10.
1 Substrate Specificity
10.1.1 Understanding of Substrate Specificity
Enzymatic Reaction Overview:
• Enzymatic reactions commence with the
binding of a substrate to the enzyme.
• As the substrate approaches the enzyme's
active site, the electrostatic microenvironment
in the substrate-binding region undergoes
changes.
• These changes facilitate and guide the
progression of the reaction toward the
formation of the final products.
Significance of Correct Substrate Binding:
• The precise and accurate binding of the
substrate to the substrate-binding site of the
enzyme is crucial.
• The specificity of an enzyme is intricately linked
to both the binding configuration and the
affinity of the substrate to the active site of the
enzyme.
Example of Substrate Specificity - Protease:
• Proteases serve as an exemplary illustration of
substrate specificity.
• Proteases encompass various types, including
trypsin, chymotrypsin, and thrombin, all of
which share the common function of cleaving
peptide bonds.
• However, they act on distinct peptide bonds,
demonstrating their specificities:
a. Trypsin:
Acts on peptide bonds involving lysine or arginine.
b. Chymotrypsin:
Acts on peptide bonds involving tyrosine, tryptophan,
or phenylalanine.
c. Thrombin:
Acts on arginine-glycine peptide bonds.
Complementarity in Enzyme-Substrate Interaction:
• Classical models explaining enzyme-substrate
reactions are founded on the concept of
complementarity between the geometric
shapes of the substrate and the enzyme's
active site.
• The substrate-binding region of an enzyme
possesses a specific geometric shape that
matches that of the substrate molecule.
• This implies that enzymes interact selectively
with compounds possessing a structurally
similar geometry.
"Lock and Key" Model:
• The specificity of enzyme-substrate interactions
is elucidated by the "Lock and Key" model.
• In this model, the enzyme corresponds to the
"lock," while the substrate represents the "key."
• Only when the key (substrate) precisely
matches the shape of the lock (active site) can
the reaction occur.
• This theory relies on the "rigid enzyme" model,
emphasizing the importance of geometric
congruence between the enzyme and the
substrate.
• The traditional "Lock and Key" model, which
assumes a rigid enzyme structure, is not always
applicable in explaining experimental
observations.
Induced-Fit Theory:
• The "Induced-Fit Theory" posits that enzymes
undergo structural alterations upon substrate
binding.
• The resulting enzyme shape is tailored to
accommodate the substrate with the correct
configuration.
• Some compounds can bind to the enzyme
without undergoing a reaction, a phenomenon
explained by this theory.
• Only specific substrates induce the appropriate
configuration of the substrate-binding site.
Predicting Substrate Specificity:
• To predict substrate specificity, an in silico high-
throughput method was suggested (Tyagi and
Pleiss, 2006).
• This method involves docking the substrate into
the enzyme, yielding transition state analogous
intermediates.
 • By analyzing side chain orientations and the
affinity of the enzyme-substrate complex,
substrate specificity can be predicted.
• This predictive approach has practical
applications, including virtual screening of
potential substrates and enzyme engineering.
10.1.2 Engineering Substrate Specificity
Enzymes in Industrial Applications:
• Increasing interest in using enzymes for
industrial chemical, biofuel, and
pharmaceutical production.
• Enzymes offer advantages in these processes,
including safety, energy efficiency, and
environmental friendliness.
Limitations of Natural Enzymes:
• Natural enzymes have finite catalytic
capabilities, often tailored for their natural
functions rather than human industrial needs.
• Natural enzymes may not be suitable for many
industrial applications or meet the
requirements of industrial biotechnology.
• Development and improvement of biosynthetic
pathways using only natural enzymes can be
hindered by limited catalytic diversity.
Engineering Enzyme-Substrate Specificity:
• Modifying the active site structure of enzymes
is essential for altering enzyme-substrate
specificity.
• Rational and computational approaches are
commonly used for this purpose.
• The key steps in the engineering process include
selecting mutation sites in the binding pocket
and then mutating selected residues to
appropriate counterparts.
Rational and Computational Approach:
• With increasing enzyme crystal data and
knowledge, rational and computational
methods, or semi-rational design, are gaining
favor.
• These methods are more favorable for
engineering enzyme-substrate specificity.
• Provide comprehensible information to
understand the structure-function
relationships involved in enzyme specificity.
Directed Evolution: A Non-Structure-Based
Approach:
• Directed evolution is a non-structure-based
protein engineering method.
• It doesn't require prior knowledge of
enzyme structure or mechanism (Arnold
and Volkov, 1999).
• Achieved through DNA shuffling or error-
prone PCR.
• Practical and effective results can be
obtained without structural information.
• However, it involves multiple rounds of
evolution and screening, making it time
and labor-intensive.
• Still widely used due to its practical
effectiveness.
Limitations of Random Approaches:
• Random approaches, like directed evolution,
don't consider the structure-function
relationships in enzymes.
• Understanding the role of applied mutations in
enzyme catalysis is challenging.
Rational Design Based on Protein Structures:
• Rational design relies on protein structure data
to propose mutations.
• These mutations can be introduced via site-
directed mutagenesis.
• Advantages of rational design:
Increases the likelihood of effective mutations.
Reduces the size of the mutation library.
Saves time and effort in library screening.
Doesn't require a high-throughput assay system,
reducing experimental costs.
Reshaping the Substrate-Binding Pocket:
• Rational design typically involves reshaping the
structure of the substrate-binding pocket.
• This is done by considering geometric and/or
physico-chemical complementarity between
the enzyme and the target substrate.
Common Strategies in Rational Design:
Strategies in the redesign process include:
Increasing the binding pocket space to
accommodate larger substrates.
 Decreasing the binding pocket space to fit
smaller substrates.
Controlling the substrate-binding mode by
adjusting molecular interactions between the
substrate and amino acid residues in the active
site (Manna and Mazumdar 2010; Mouratou et
al. 1999; Sinclair et al. 1998).
Reversible Inhibitors:
• Reversible inhibitors bind to the enzyme but
can dissociate from it relatively easily.
• This binding does not permanently damage the
enzyme, allowing for activity recovery upon
inhibitor dissociation.

Qualitative Nature of Rational Design: Irreversible Inhibitors:

• Rational design, despite being based on
structural information, is a qualitative
approach.
• It heavily relies on the insight and expertise of
researchers.
• Primarily focuses on the "binding"
characteristics of enzymes.
Semi-Rational Approach Overview:
• Combines elements of both rational and
random approaches for enzyme improvement.
• Utilizes saturation mutagenesis on the
enzyme's active site.
• Widely employed for designing enzyme-
substrate specificity (Andrews and McLeish
2013; Gao et al. 2013; Xie et al. 2014).
Focused Mutagenesis on Active Site:
• Unlike the entirely random approach, semi-
rational approach concentrates on the active
site.
• This targeted mutagenesis enhances the
efficiency of the design process.
• Irreversible inhibitors, such as mercury and
lead, form strong and lasting bonds with the
enzyme's amino acid backbone.
• This binding leads to a time-dependent loss of
enzyme activity, and recovery is usually not
possible.
Types of Inhibition:
There are three primary types of enzyme inhibition:
Competitive Inhibition: Inhibitor competes with
the substrate for the active site on the enzyme.
Noncompetitive Inhibition: Inhibitor binds to a
different site on the enzyme, altering its
conformation and reducing substrate binding.
Uncompetitive Inhibition: Inhibitor only binds
to the enzyme-substrate complex, preventing
the release of the product.
Models for more complex enzyme kinetics

Synergistic Effects:

• Semi-rational approach capitalizes on the

strengths of both rational and random
approaches.
• Pragmatically efficient for enhancing enzyme
variants.

4.2.1 Inhibition Kinetics

Enzyme Inhibitors:

• Enzyme inhibitors are compounds that reduce

the rate of an enzyme reaction.
• The loss of enzyme activity can be either
reversible or irreversible.