Enzymes

1
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

2
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.

3
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.

4
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.

5
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.

6
Starch Conversion High Fructose Corn Syrup

• a-amylase degrades amylose to D-glucose, 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 low-cost (relative to sucrose) natural
  sweetener in soft drinks

7
Starch Conversion Isomerization
8
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
• 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
• from 60 to 1000 are
• known.

9
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 (RH), the
• a-amino acids have at least one
• asymmetric carbon atom that exists in
• the S-configuration.
• 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.

10
Common a-Amino Acids
11
Common a-Amino Acids
12
Structure of Enzymes

• Enzymes have genetically mandated and unique
  amino acid sequences
• 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.

13
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.
• However, peptide bonds
• alone do not define primary
• structure, as disulfide bonds
• between cysteine residues
• crosslink different parts of the
  peptide chain.

14
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

15
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.

16
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

17
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.

18
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.

19
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.

20
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.