Download L16-Enzyme Structure

Enzymes
Enzymes are catalytically active proteins that regulate virtually all
biological processes. Industrial applications can be found in:
 Fine chemical preparations
 Food-industry (dairy, starch conversion)
 Analytical chemistry and medicine
While the activity of enzyme-mediated reactions is exceptional, it is
selectivity of these processes that is both unique and valuable:
 Reaction specificity
 Regioselectivity: yields one of several structural isomers
 Stereoselectivity: consumes/yields one stereoisomer
(enantiomer)
Topics for Discussion
 Enzyme production and purification schemes
 Industrial applications
 Enzyme structure and the nature of the catalytic site
 Catalytic chemistry and reaction kinetics
 Enzyme immobilization and mass transfer
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Enzyme Production/Isolation Methods
The structural complexity of enzymes
makes their synthetic preparation a
formidable task. They are natural
products that are isolated/produced from
three principle sources.
 Isolation from animal organs (hog
insulin)
 Isolation from plant material
(papain)
 Microorganism production
Isolation and purification are complicated
by the presence of similar proteins and
the inherent sensitivity of enzymes to
pH, temperature and degradation by
other enzymes.
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Classification of Enzymes
I. Oxidoreductases: encompass all enzymes that catalyze redox reactions.
Name is dehydrogenase whenever possible, but reductase can also be
used. Oxidase is used only where 0 is the acceptor for reduction.
2. Transferases: catalyze the transfer of a specific group such as methyl,
amino or phosphate from one substance to another. Name is acceptor
group- transferase or donor group-transferase.
3. Hydrolases: catalyze the hydrolytic cleavage of C-O, C-N, C-C, and some
other bonds. Name often consists of the substrate name with the suffix -ase.
4. Lyases: catalyze the cleavage of C-C, C-O, C-N, and other bonds by
elimination. Name is, for example, decarboxylase, dehydratase (elimination
of CO and water, respectively).
5. Isomerases: catalyze geometric or structural rearrangements within a
molecule. Different types lead to the names racemase, epimerase,
isomerase, tautomerase, or cycloisomerase.
6. Ligases: catalyze the joining of two molecules, coupled with the hydrolysis
of a pyrophosphate bond in ATP or another nucleoside triphosphate.
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Applications of Proteolytic Enzymes
Dairy:
Calf rennet (chymosin) is used in the coagulation of milk protein for
cheese production, without loss of sensitive components.
Lactase hydrolyzes the principal carbohydrate of milk, lactose. This
processes a cheese byproduct and relieves lactose intolerance.
Detergents:
Protein stain removal is facilitated by the hydrolysis of proteins into
oligopeptides. Enzyme stability with respect to storage, pH,
temperature and bleach are key concerns.
Leather Production:
Proteases are widely used for the soaking and dewooling stages of
hide processing in which selective protein degradation results in a
softer produce without substantial loss of strength.
Food and Feed:
Starch conversion to high-fructose corn syrup is an important
process to the beverage industry.
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Starch Conversion
The processing of starch to yield sweeteners is a significant
industrial operation. Acid-catalyzed hydrolysis has been largely
supplanted by enzyme-mediated hydrolysis due to superiour
activity and reduced by-product formation.
Starch contains two polysaccharides, amylose and amylopectin.
Amylose (n approx. 400)
amylopectin
(branched)
Hydrolysis of amylose yields the constituent monomer D-glucose,
while the degradation of amylopectin is complicated by its
branched structure.
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Starch Conversion: High Fructose Corn Syrup
a-amylase degrades amylose to Dglucose, but a second enzyme,
glucoamylase is needed to breakdown
oligosaccharides derived from
amylopectin.
This product can be used as substrate for
yeast fermentation to produce ethanol as
an alternate fuel source. Much of the
glucose produced by starch degradation is
isomerized to fructose for use as a lowcost (relative to sucrose) natural
sweetener in soft drinks
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Starch Conversion: Isomerization
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Enzyme “Building-Blocks”: a-Amino Acids
An enzyme is a singular macromolecule with precise monomer
sequencing. As with all proteins, the monomers that constitute
enzymes are a-amino acids:
R
C
H
H2N
R
CO2H
C
H
CO2H3N +
The zwitterionic character results from the potential for proton
donation from the carboxylic acid group to the basic amino
functionality of the molecule.
Condensation of amino acids yields biological oligomers/polymers
known as peptides, which if catalytically active are enzymes.
 Degrees of polymerization
2
O
R
O
from 60 to 1000 are
NH
NH
known.
NH
R
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O
R
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Enzyme “Building-Blocks”: a-Amino Acids
There are twenty amino acids that occur commonly as constituents
of most proteins. With but one exception (proline) all a-amino
acids have the same general structure, differing only in the
substituent R.
With the exception of glycine (R=H), the
a-amino acids have at least one
asymmetric carbon atom that exists in
the S-configuration.
R
C
H
H2N
CO2H
The backbone structure of peptides derived from the a-amino
acids is capable of hydrogen-bonding to yield highly ordered chain
conformations.
The substituents, R, range from non-polar aliphatics and aromatics
to polar alcohols, amines and carboxylic acids.
 The nature of a substituent affects enzyme conformation as
well as the chemistry by which a reaction is catalyzed.
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Common a-Amino Acids
H O
+
H3N C C O
R
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Common a-Amino Acids
H O
+
H3N C C O
R
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Structure of Enzymes
Enzymes have genetically mandated and unique amino acid
sequences:
2
NH
O
R
O
NH
NH
R
1
O
R
3
Although only a small subset of the amino acids within an enzyme
may engage the reactant(s), all enzyme constituents are needed
for catalytic activity. Enormous molecule size generates:
 sufficient local-controlled flexibility
 precise three dimensional arrangements
In spite of the tremendous structural complexity of enzymes,
reactions derived from their reactive functional groups are similar to
the acid-base and metal-mediated processes you have already
studied.
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Primary Structure of Enzymes
The complex structure of enzymes can
be discussed at different levels, the
simplest of which is the covalent
structure, or primary structure.
The most important aspect is the
amino acid sequence, shown here
for lysozyme.
R
1
NH
NH2
CH2 S
O
NH
NH
CH2 S
O
3
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R
polypeptide
chain
2
disulfide bond between
two cysteine residues
O
NH
NH
R
O
O
O
R
4
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However, peptide bonds
alone do not define primary
structure, as disulfide bonds
between cysteine residues
crosslink different parts of the
peptide chain.
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Secondary Structure of Enzymes
Macromolecules which lack polar functional groups assume random
coil configurations in solution that are dictated by polymer-solvent
interactions.
Peptides on the other hand have
restricted rotation about the
carbonyl-nitrogen bond of the amide
linkage, thereby “locking” one site of
potential backbone flexibility into
the Z-conformation.
The description of enzyme structure in terms of ordered domains is
referred to as the secondary structure. Three peptide conformations
are most commonly assumed:
 random coil conformation
 a-helix
 b-pleated sheet
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Secondary Structure of Enzymes
A common peptide backbone
configuration is a right-handed
a-helix.
A helical conformation of the
enzyme is generated through
hydrogen-bond interactions
between the amide N-H of
one residue and the carbonyl
oxygen four residues away.
The side-chain groups are
positioned on the outside of
the helix.
R
O
NH
2
O
NH
NH
R
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O
R
3
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Secondary Structure of Enzymes
A b-structure, or pleated sheet is generated by hydrogen-bonding
interactions between peptide chains (or a different part of the same
chain).
 A peptide chain adopts an open, zigzag conformation
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Tertiary Structure of Enzymes
The complete three dimensional structure of a protein is called its
tertiary structure. It is an aggregate of a-helix, b-sheet and random
coil and other structural elements that is governed by non-covalent
interactions.
While covalent (peptide and disulfide
bonds) interactions define the primary
structure, non-covalent interactions
determine the tertiary structure
of proteins:
 Hydrogen bonding
 Van der Waals interactions
 Electrostatic interactions
Note that amino acid sequencing, as
defined by the primary structure,
determines the extent of these non-covalent interactions, and
defines the secondary and tertiary structure of the enzyme.
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Tertiary Structure of Enzymes
In solution an enzyme adopts a
lowest-energy conformation which,
owing to the uniqueness of the
primary structure, is very precise.
The polarity of the amino acid
functional groups dictates the
affinity of particular peptide
sequences for water. Hydrophilic
sequences favour positions on the
surface of the enzyme, while
hydrophobic sequences are found
in the internal regions of the protein.
 This behaviour is analgous to
micelle formation in
surfactants.
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Quaternary Structure of Enzymes
Many enzymes consist of more than one polypeptide chain (or
subunit) that aggregate to confer catalytic activity.
 In some enzymes the subunits are identical, in other cases
they differ in sequence and structure.
 The description of subunit arrangement in such enzymes is
called the quaternary structure.
A typical enzyme is not an entity completely folded as a whole, but
may consist of apparently autonomous or semi-autonomous folding
units called domains.
 Functional domains (those providing catalytic activity) can be
regions that fold independently
 The active site of lysozyme (slide 11) is believed to be the
cleft between two distinct domains.
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Summary of Enzyme Structure
Enzymes are catalytically active macromolecules comprised of a specific
sequence of a-amino acids.
 The sequence of amino acids through peptide bonds and chain
crosslinking through disulfide bonds of cysteine residues is the
primary structure of the enzyme.
 Peptide sequences can form ordered subunits through hydrogen
bonding interactions. These include the a-helix, b-sheet. Random
coil conformations predominate in the remaining peptide sequences.
These comprise the secondary structure of the enzyme.
 Non-covalent interactions between the elements of the secondary
structure generate the very specific overall conformation of the
enzyme, called the tertiary structure.
 Where more than one peptide chain aggregates to generate the
active enzyme, a quaternary structure is defined.
The structure of an enzyme represents the lowest-energy conformation of
the macromolecule, which will spontaneously form given the appropriate
primary structure.
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