ENZYMES

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ENZYMES

• Medical Biochemistry, Lecture 23

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Lecture 23, Outline

• Definition of enzyme terms and nomenclature
• Description of general properties of enzymes
• Binding energy and transition states
• Catalytic mechanisms and functional groups
• In book, Chapters 8,10 Ignore pp 78-80
• Recommended supplement for lectures 23-25
  UNDERSTAND Biochemistry CD

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Enzyme Catalysis Overview
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Enzyme Nomenclature

• active site - a region of an enzyme comprised of
  different amino acids where catalysis occurs
  (determined by the tertiary and quaternary
  structure of each enzyme)
• substrate - the molecule being utilized and/or
  modified by a particular enzyme at its active
  site
• co-factor - organic or inorganic molecules that
  are required by some enzymes for activity. These
  include Mg2, Fe2, Zn2 and larger molecules
  termed co-enzymes like nicotinamide adenine
  dinucleotide (NAD), coenzyme A, and many
  vitamins.

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Enzyme Nomenclature (cont)

• prosthetic group - a metal or other co-enzyme
  covalently bound to an enzyme
• holoenzyme - a complete, catalytically active
  enzyme including all co-factors
• apoenzyme - the protein portion of a holoenzyme
  minus the co-factors
• isozyme - (or iso-enzyme) an enzyme that performs
  the same or similar function of another enzyme.
  This generally arises due to similar but
  different genes encoding these enzymes and
  frequently is tissue-type specific or dependent
  on the growth or developmental status of an
  organism.

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Clinical Use of Enzymes

• Enzyme Activity in Body Fluids Reflects Organ
  Status
• Cells die and release intracellular contents
  increased serum activity of an enzyme can be
  correlated with quantity or severity of damaged
  tissues (ex. creatine kinase levels following
  heart attack)
• Increased enzyme synthesis can be induced and
  release in serum correlates with degree of
  stimulation (ex. alkaline phosphatase activity as
  a liver status marker)

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Clinical Use of Enzymes (cont)

• Enzyme Activity Reflects the Presence of
  Inhibitors or Activators
• Activity of serum enzymes decreases in presence
  of an inhibitor (ex. some insecticides inhibit
  serum cholinesterases)
• Determine co-factor deficiencies (like an
  essential vitamin) by enzyme activity (ex. add
  back vitamin to assay, if activity increases,
  suggests deficiency in that vitamin)

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Clinical Use of Enzymes (cont)

• Enzyme activity can be altered genetically
• A mutation in an enzyme can alter its substrate
  affinity, co-factor binding stability etc. which
  can be used as a diagnostic in comparison with
  normal enzyme
• Loss of enzyme presence due to genetic mutation
  as detected by increased enzyme substrate and/or
  lack of product leading to a dysfunction
• NOTE PCR techniques that identify specific
  messenger RNA or DNA sequences are replacing many
  traditional enzymatic based markers of genetic
  disease

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ENZYMATIC REACTION PRINCIPLES

• Biochemically, enzymes are highly specific for
  their substrates and generally catalyze only one
  type of reaction at rates thousands and millions
  times higher than non-enzymatic reactions. Two
  main principles to remember about enzymes are 1)
  they act as CATALYSTS (they are not consumed in a
  reaction and are regenerated to their starting
  state) and 2) they INCREASE THE RATE of a
  reaction towards equilibrium (ratio of substrate
  to product), but they do not determine the
  overall equilibrium of a reaction.

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CATALYSTS

• A catalyst is unaltered during the course of a
  reaction and functions in both the forward and
  reverse directions. In a chemical reaction, a
  catalyst increases the rate at which the reaction
  reaches equilibrium, though it does not change
  the equilibrium ratio. For a reaction to proceed
  from starting material to product, the chemical
  transformations of bond-making and bond-breaking
  require a minimal threshold amount of energy,
  termed activation energy. Generally, a catalyst
  serves to lower the activation energy of a
  particular reaction.

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ENZYMATIC REACTION PRINCIPLES (cont)

• The energy maxima at which the reaction has the
  potential for going in either direction is termed
  the transition state. In enzyme catalyzed
  reactions, the same chemical principles of
  activation energy and the free energy changes
  (DGo) associated with catalysts can be applied.
  Recall that an overall negative DGo indicates a
  favorable reaction equilibrium for product
  formation. As shown in an enzyme catalyzed
  reaction, and in the graph, the net effect of the
  enzyme is to lower the activation energy required
  for product formation.

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Chemical Reaction
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Enzymatic Reaction Energetics
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Reaction Rates

• The rate of the reaction is determined by several
  factors including the concentration of substrate,
  temperature and pH. For most standard
  physiological enzymatic reactions, pH and
  temperature are in a defined environment (pH
  6.9-7.4, 37oC). Therefore, the concentration of
  substrate is the critical determinant. This
  enzymatic rate relationship has been described
  mathematically by combining the equilibrium
  constant (the ratio of substrate and product
  concentrations), the free energy change and first
  or second-order rate theory. The net result for
  enzymatic reactions is that the lower the
  activation energy, the faster the reaction rate,
  and vice versa.

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Binding Energy

• The graph of activation energy and free energy
  changes for an enzymatic reaction also indicates
  the role binding energy plays in the overall
  process. Due to the high specificity most
  enzymes have for a particular substrate, the
  binding of the substrate to the enzyme through
  weak, non-covalent interactions is energetically
  favorable and is termed binding energy. The same
  forces important in stabilizing protein
  conformation (hydrogen bonding and hydrophobic,
  ionic and van der Waals interactions) are also
  involved in the stable binding of a substrate to
  an enzyme.

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Binding Energy and Transition State

• The cumulative binding energies from the
  non-covalent interactions are optimized in the
  transition state and is the major source of free
  energy used by enzymes to lower activation
  energies of reactions. A single weak interaction
  has been estimated to yield 4-30 kJ/mol energy,
  thus multiple interactions (which generally would
  occur during binding and catalysis) can yield up
  to 60-80 kJ/mol free energy - this accounts for
  the large decreases in activation energies and
  increases in rate of product formation observed
  in enzymatic-catalyzed reactions.

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Effect of Temperature
A reaction rate will generally increase with
increasing Temperature due to increased kinetic
energy in the system until a maximal velocity is
reached. Above this maximum, the kinetic energy
of the system exceeds the energy barrier for
breaking weak H-bonds and hydrophobic
interactions, thus leading to unfolding and
denaturation of the enzyme and a decrease in
reaction rate.
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Effect of pH
Variations in pH can affect a particular enzyme
in many ways, especially if ionizable amino acid
side chains are involved in binding of the
substrate and/or catalysis. Extremes of pH can
also lead to denaturation of an enzyme if the
ionization state of amino acid(s) critical to
correct folding are altered. The effects of pH
and temperature will vary for different enzymes
and must be determined experimentally.
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LOCK-AND- KEY
INDUCED FIT
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Hexokinase Active Site Glucose vs. Galactose
Binding
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Co-factor NAD/NADH
(EXAMPLE)
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Co-factors Co-A and Biotin
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Catalytic Mechanisms Types

• Four types of catalytic mechanisms will be
  discussed
• binding energy catalysis
• general acid-base catalysis
• covalent catalysis
• metal ion catalysis

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Acid-Base Catalysis
Many reactions involve the formation of normally
unstable, charged intermediates. These
intermediates can be transiently stabilized in
an enzyme active site by interaction of amino
acid residues acting as weak acids (proton
donors) or weak bases (proton acceptors). The
general acid and general base forms of the most
common and best characterized amino acids
involved in these reactions are shown above.
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Acid-Base Catalysis (cont)

• The preceding functional groups can potentially
  serve as either proton donors or proton
  acceptors. This is dependent on many factors
  including the molecular nature of the substrate,
  any co-factors involved, and the pH of the active
  site (which would determine the ionization state
  of an amino acid side chain). For acid-base
  catalysis, histidine is the most versatile amino
  acid due to its pKa which means that in most
  physiological situations it can act as either a
  proton donor or proton acceptor. Generally these
  amino acids will interact together with the
  substrate, or in conjunction with water or other
  weak, organic acids and bases found in cells.

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Binding Energy Catalysis

• Binding energy accounts for the overall lowering
  of activation energy for a reaction, and it can
  also be considered as a catalytic mechanism for a
  reaction. Several catalytic factors in the
  binding of a substrate and enzyme can be
  considered 1) transient limiting of substrate
  and enzyme movement by reducing the relative
  motion (or entropy) of the two molecules, 2)
  solvation disruption of the water shell is
  thermodynamically favorable, and 3) substrate and
  enzyme conformational changes. All three of these
  factors individually or in combination are
  utilized to some degree by an enzyme. While in
  some instances these forces alone can account for
  catalysis, they are frequently components of a
  complex catalytic process involving factors
  discussed for the other types of catalytic
  mechanisms.

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Covalent Catalysis

• This mechanism involves the transient covalent
  binding of the substrate to an amino acid residue
  in the active site. Generally this is to the
  hydroxyl group of a serine, although the side
  chains of threonine, cysteine, histidine,
  arginine and lysine can also be involved.

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Metal Ion Catalysis

• Various metals, all positively charged and
  including zinc, iron, magnesium, manganese and
  copper, are known to form complexes with
  different enzymes or substrates. This
  metal-substrate-enzyme complex can aid in the
  orientation of the substrate in the active site,
  and metals are known to mediate
  oxidation-reduction reactions by reversible
  changes in their oxidation states (like Fe3 to
  Fe2).

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Summary of Catalytic Mechanisms

• In general, more than one type of catalytic
  mechanism will occur for a particular enzyme via
  various combinations of binding energy,
  acid-base, covalent and metal catalysis. Enzymes
  as a whole are incredibly diverse in their
  structures and the types of reactions that they
  catalyze, therefore there is also a large
  diversity of catalytic mechanisms utilized, the
  basis of which must be determined experimentally.

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Substrate Binding Pockets of Chymotrypsin and
Trypsin
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Catalytic Mechanism of Chymotrypsin
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Chymotrypsin Mechanism (cont)
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Chymotrypsin, last step and regeneration of
active enzyme