ENZYME SPECIFICITY

 Enzyme specificity refers to the tendency of the enzymes to catalyze only a specific set of chemical
reactions with the ligand as a substrate.
 It is one of those properties of enzymes that makes them so important as diagnostic and research tools.
 Specificity describes the strength of binding between a given protein and ligand.
 The fewer ligands a protein can bind, the greater its specificity.
 If a given enzyme has a high chemical specificity, this means that the set of ligands to which it binds is
limited, such that neither binding events nor catalysis can occur at an appreciable rate with additional
molecules.
 An example of a protein-ligand pair whose binding activity can be described as highly specific is
the antibody-antigen system.
 Conversely, an example of a protein-ligand system that can bind substrates and catalyze multiple
reactions effectively is the Cytochrome P450 system, which can be considered a promiscuous
enzyme due to its broad specificity for multiple ligands.
o Cytochromes P450 (CYPs) are a family of enzymes containing heme as a cofactor that
function as monooxygenases.
o In mammals, these proteins oxidize steroids, fatty acids, and xenobiotics, and are
important for the clearance of various compounds, as well as for hormone synthesis and
breakdown.
o In plants, these proteins are important for the biosynthesis of defensive compounds, fatty
acids, and hormones.
 The relationship between an enzyme and the ligand can be described by the dissociation constant,
that characterizes the balance between bound and unbound states for the protein-ligand system.
 In the context of a single enzyme and a pair of binding molecules, the two ligands can be
compared as stronger or weaker ligands (for the enzyme) on the basis of their dissociation
constants. (A lower value corresponds to a stronger binding.)
 So the question arises is “What determines the specificity of an enzyme for its substrate?”
 To which the answer is Active site of an enzyme. For a substrate to bind to the active site of an
enzyme it must fit in the active site and be chemically attracted to it.
 Active Site: region of an enzyme where substrate molecules bind and undergo a chemical
reaction.
 It consists of residues that form temporary bonds with the substrate (binding site) and residues
that catalyse a reaction of that substrate (catalytic site).

 The interactions between the protein and ligand substantially affect the specificity between the two
entities.
 Electrostatic interactions and Hydrophobic interactions are known to be the most influential in
this regards.
 In order to carry out various physiological functions, enzymes vary in the specificity of the substrates
that they bind to.
 While some enzymes may need to be less specific, certain physiological functions require extreme
specificity of the enzyme for a single specific substrate.
 The different types of categorizations differ based on their specificity for substrates.
 Most generally, they are divided into four groups: absolute, group, linkage, and stereochemical
specificity.
Absolute specificity
 Absolute specificity can be thought of as being exclusive, in which an enzyme acts upon one
specific substrate.
 Absolute specific enzymes will only catalyze one reaction with its specific substrate.
 For example, lactase is an enzyme specific for the degradation of lactose into two sugar
monosaccharides, glucose and galactose.
 Another example is Glucokinase, which is an enzyme involved in the phosphorylation of
glucose to glucose-6-phosphate. It is primarily active in the liver and is the main isozyme of
Hexokinase.
 Its absolute specificity refers to glucose being the only hexose that is able to be its substrate, as
opposed to hexokinase, which accommodates many hexoses as its substrate.
Group specificity
 Group specificity occurs when an enzyme will only react with molecules that have specific
functional groups, such as aromatic structures, phosphate groups, and methyls.
 One example is Pepsin, an enzyme that is crucial in digestion of foods ingested in our diet, that
hydrolyzes peptide bonds in between hydrophobic amino acids, with recognition for aromatic
side chains such as phenylalanine, tryptophan, and tyrosine.
 Another example is hexokinase, an enzyme involved in glycolysis that phosphorylate glucose
to produce glucose-6-phosphate. This enzyme exhibits group specificity by allowing multiple
hexoses (6 carbon sugars) as its substrate.
 Glucose is one of the most important substrates in metabolic pathways involving hexokinase
due to its role in glycolysis, but is not the only substrate that hexokinase can catalyze a reaction
with.
Bond specificity
 A reaction that illustrates an enzyme cleaving a specific bond of the reactant in order to create
two products.
 Bond specificity, unlike group specificity, recognizes particular chemical bond types.
 This differs from group specificity, as it is not reliant on the presence of particular functional
groups in order to catalyze a particular reaction, but rather a certain bond type (for example, a
peptide bond).
Stereochemical specificity
 This type of specificity is sensitive to the substrate’s optical activity of orientation.
 Stereochemical molecules differ in the way in which they rotate plane polarized light, or
orientations of linkages (see alpha, beta glycosidic linkages).
 Enzymes that are stereochemically specific will bind substrates with these particular properties.
 For example, beta-glycosidase will only react with beta-glycosidic bonds which are present in
cellulose, but not present in starch and glycogen, which contain alpha-glycosidic linkages.
 This is relevant in how mammals are able to digest food.
 For instance, the enzyme Amylase is present in mammal saliva, that is stereo-specific for alpha-
linkages, this is why mammals are able to efficiently use starch and glycogen as forms of
energy, but not cellulose (because it is a beta-linkage).

APPLICATION TO ENZYME KINETICS

The chemical specificity of an enzyme for a particular substrate can be found using two variables
that are derived from the Michaelis-Menten equation.
km approximates the dissociation constant of enzyme-substrate complexes.
kcat represents the turnover rate, or the number of reactions catalyzed by an enzyme over the
enzyme amount.
o Specificity constant, gives a measure of the affinity of a substrate for a particular enzyme.
o Denotes the efficiency of an enzyme, this relationship reveals an enzyme's preference for a
particular substrate.
o The higher the specificity constant of an enzyme corresponds to a high preference for that
substrate.
IMPORTANCE TO MEDICAL RESEARCH
 Specificity is important for novel drug discovery and the field of clinical research, with new
drugs being tested for its specificity to the target molecules.
 Drugs must contain as specific as possible structures in order to minimize the possibility of off-
target affects that would produce unfavorable symptoms in the patient.
 Drugs depend on the specificity of the designed molecules and formulations to inhibit particular
molecular targets.
 Novel drug discovery progresses with experiments involving highly specific compounds.
o For example, the basis that drugs must successfully be proven to accomplish is both the
ability to bind the target receptor in the physiological environment with high specificity
and also its ability to transduce a signal to produce a favorable biological effect against the
sickness or disease that the drug is intended to negate.
 Though enzymes exhibit great degrees of specificity, cofactors may serve many apoenzymes.
o For example, nicotinamide adenine dinucleotide (NAD) is a coenzyme for a great number
of dehydrogenase reactions in which it acts as a hydrogen acceptor. Among them are the
alcohol dehydrogenase, malate dehydrogenase and lactate dehydrogenase reactions.
 ACTIVE SITE
 The active site is the region of an enzyme where substrate molecules bind and undergo a
chemical reaction.
 It consists of residues that form temporary bonds with the substrate (binding site) and residues
that catalyse a reaction of that substrate (catalytic site).
 As compared to the overall enzymatic structure, the active site is relatively small and only
occupies 10~20% of the total volume.
 However, it is the most important part of the enzyme as it directly catalyzes the chemical
reaction.
 It usually consists of three to four amino acids, while other amino acids within the protein are
required to maintain the protein tertiary structure of the enzyme.
 Each active site is evolved to be optimised to bind a particular substrate and catalyse a particular
reaction, resulting in high specificity.
 This specificity is determined by the arrangement of amino acids within the active site and the
structure of the substrates.
 Since the residues aren’t altered on the completion of the reaction, an active site can catalyse a
reaction repeatedly (as per the induced fit theory, they may change during the reaction, but are
regenerated by the end). This process is achieved by lowering the activation energy of the
reaction, so more substrates have enough energy to undergo reaction.
 An active site contains a binding site that binds the substrate and orients it for catalysis.
 Initially, the interaction between the active site and the substrate is non-covalent and transient.
 There are four important types of interaction that hold the substrate in a defined orientation and
form an enzyme-substrate complex (ES complex): hydrogen bonds, van der Waals interactions,
hydrophobic interactions and electrostatic force interactions.
 The charge distribution on the substrate and active site must be complementary, which means all
positive and negative charges must be cancelled out. Else there will be a repulsive force pushing
them apart.
 The active site usually contains non-polar amino acids, although sometimes polar amino acids
may also occur.
 Once the substrate is bound and oriented to the active site, catalysis can begin. The residues of
the catalytic site are typically very close to the binding site, and some residues can have dual-
roles in both binding and catalysis.
 Catalytic residues of the site interact with the substrate to lower the activation energy of a
reaction and thereby make it proceed faster. They do this by a number of different mechanisms
including the approximation of the reactants, nucleophilic/electrophilic catalysis and acid/base
catalysis.