Enzymes

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Enzymes

• Two fundamental conditions for life are that 1) a
  living entity must be able to self-replicate, and
  2) a living entity must be able to catalyze
  chemical reactions efficiently and selectively.
• Almost all biochemical processes are catalyzed by
  a highly specialized class of proteins, called
  enzymes.
• Enzymes have a high degree of specificity for
  their substrates.
• Enzymes accelerate chemical reactions
  tremendously.
• Enzymes can function in aqueous solution under
  mild conditions, which are unlike the conditions
  that are frequently needed in organic chemistry.
• Almost all Enzymes are proteins. Their ability
  to catalyze reactions is attributable to their
  primary, secondary, tertiary, and quaternary
  structures.
• Reading. Lehninger Chap 1, pp. 11-12, Chap 3,
  pp. 64-68, Chap 8, pp. 243-288.

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Prosthetic Groups

• Some enzymes require cofactors or prosthetic
  groups for their function. These typically are
  small organic molecules or metals that bind to
  the protein, and that enable the protein to carry
  out its function.
• An enzyme that lacks its cofactors or prosthetic
  groups is called an apo-enzyme. An enzyme that
  contains its cofactors or prosthetic groups is
  called a holo-enzyme.
• Vitamins typically are precursors to enzyme
  cofactors, which explains their necessity in the
  diet. Cofactors from vitamins are called
  coenzymes. Youll learn more about the function
  of vitamins in BMB 402.

3
Catalysis

• Many reactions in biochemistry are spontaneous,
  meaning that they are thermodynamically favorable
  (DGlt0). This does not mean, however, that they
  proceed rapidly.
• The oxidation of glucose to yield carbon dioxide
  and water is thermodynamically favorable
  (DG-2870 kJ/mol). However, a jar of sugar,
  even in water, is incredibly stable, and has a
  half-life that is probably in the thousands of
  years in the absence of microbial contamination.
  
• Biological reactions are almost always under
  kinetic control, in which a given amount of
  energy must be put into the system (energy of
  activation) in order for energy to be released.
  The maximum point along the reaction coordinate
  is called the transition state for the reaction.
  At this point, the activated complex can break
  down to form products, or revert back to
  reactants. The rate of a reaction is related
  exponentially to the energy of activation.
• Two ways to effect the reaction. Raise the free
  energy of the substrates, or decrease the energy
  of activation for the reaction.
• Enzymes catalyze reactions by lowering the
  activation energy barrier.

HOH Cl HO  H Cl
HO HCl
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The Michaelis Complex

• In an enzymatic reaction, the reactants are
  called substrates. Intimate interaction between
  an enzyme and its substrates occurs through
  molecular recognition based on structural and
  electrostatic complementarity. Frequently, the
  terms lock and key are used.
• The specific location on the enzyme that is
  complementary to the substrate and wherein
  catalysis takes place is called the active site.
  
• The binding of the substrate to the enzyme
  results in formation of the enzymesubstrate
  complex, termed the Michaelis complex, or simply
  the ES complex.

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Catalysis

• Enzymes cannot change the equilibrium constant of
  any particular reaction, they can only speed the
  onset to equilibrium.
• The energy barrier between S and P, is called the
  activation energy, which is that required to
  reach the transition state. This energy reflects
  the formation of transient unstable charges, bond
  rearrangements, the alignment of reacting groups,
  and other transformations that are necessary for
  the reaction to proceed.
• The rate of a reaction correlates with the
  activation energy. The higher the activation
  energy (more unstable the transition state), the
  slower the reaction.

E S ? ES ? EP ? E P
This reaction proceeds spontaneously in the S to
P direction.
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The Importance of Binding Energy

• The active sites of enzymes tend to be more
  complementary to the transition states of their
  respective reactions than they are to the actual
  substrates.
• This results in lowering the energy of the
  enzymetransition state complex, meaning, a
  lowering of the activation energy.
• In order for catalysis to be effective, the
  energy barrier between ES and EXt must be less
  than S and Xt.
• Notice that the binding of substrate to enzyme
  lowers the free energy of the ES complex relative
  to substrate. If the energy is lowered too much,
  without a greater lowering of EXt, then catalysis
  would not take place.
• Notice how the magnetic interactions (binding
  determinants) compensate for the energy required
  to bend the stick (substrate)

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Binding Energy and Entropy Loss

• How is it that the transition state can be
  stabilized more than the substrate at the enzyme
  active site?
• The favorable interactions between the substrate
  and amino acid residues on the enzyme account for
  the intrinsic binding energy DGb.
• Its necessary to compensate for some of this
  binding energy. There is also an energy of
  destabilization (positive value), which can arise
  from strain on the substrate in the active site,
  desolvation of the substrate and its positioning
  in a somewhat unfavorable environment.
• Compensation also occurs via the negative entropy
  that arises upon immobilization of the substrate
  in the active site.

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Entropy Loss and Destabilization of the ES Complex

• Entropy loss arises from the fact that the ES
  complex is a highly organized entity. There is a
  great degree of order in the complex.
• Translational movement as well as rotational
  movement is usually greatly restricted, giving
  rise to loss in both translational and rotational
  entropy.
• Since DS will be negative (loss in entropy), the
  TDS term will be positive, and the DG will be
  more positive, meaning less favorable.

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Catalysis and Destabilization of ES
Strain, desolvation, electrostatic repulsion
Usually the transition state is not subject to
the same degree of destabilization.
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Nature of the Transition State

• Let ke be the rate constant for an enzyme
  catalyzed reaction. Let ku be the rate constant
  for the same reaction uncatalyzed.
• Let KS ES / ES
• Let KT EXt / EXt
• KS is therefore the dissociation constant for the
  enzyme-substrate complex, and KT is the
  dissociation constant for the enzyme-transition
  state complex.
• ke / ku is about equal to KS / KT. May not seem
  intuitively obvious. Remember that we defined
  dissoication constants rather than association
  constants.
• Greater the binding to the transition state, the
  greater the rate enhancement.

Transition state analogs allow for the testing of
transition state theory. Look at the
interconversion of L-proline and D-proline,
catalyzed by proline racemase. The transition
state is presumed to be a planar-like species,
with psp2 hybridization. Two analogs of the
transition state were synthesized and shown to
bind much tighter to the enzyme than the
substrates.
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Other Transition State Analogs

• Two other examples of transition state analogs
  that bind tighter to the enzyme than the
  substrate.
• Yeast aldolase catalyzes a reversible aldol
  condensation of dihydroxyacetone phosphate and
  glyceraldehyde 3-phosphate to give
  fructrose-1,6-bisphosphate.,
• Adenosine deaminase catalyzes the hydrolysis of
  the enamine to form the modified nucleotide,
  inosine. Remember what youve learned about
  imines and their ability to be hydrolyzed.
• The Ki is simply the dissociation constant for an
  inhibitor. Km is the substrate concentration
  that gives 1/2 maximal velocity.

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Strategies in Enzyme Catalysis

• As stated earlier, the role of a catalyst is to
  decrease the energy of activation of a
  reactionthe energy necessary to attain the
  transition state.
• Several themes recur in enzyme catalysis.
• Catalysis by approximation
• General acid, general base catalysis
• Catalysis by electrostatic effects
• Covalent catalyis (nucleophilic or electrophilic)
• Catalysis by strain or distortion
• For most enzymes, more than one of these
  strategies are used concomitantly

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Enzyme Classification

• Most enzymes are named according to the function
  that they carry out with the appended suffix
  -ase.
• DNA polymerase catalyzes the polymerization of
  dexoynucleotides to form DNA.
• Lipoyl transferase catalyzes the transfer of the
  lipoyl moiety from one protein to another.
• Cyclopropane fatty acid synthase, catalyzes the
  synthesis of cyclopropane rings on fatty acids
  that comprise the membranes of certain bacteria.
• Cysteine desulfurase catalyzes the liberation of
  sulfur from the amino acid cysteine.
• Some enzymes that have been known since before a
  systematic means of nomenclature arose are still
  called by their trivial names, such as trypsin,
  pepsin, chymotrypsin, etc.
• Still, a numeric system of nomenclature hs been
  established. For example the enzyme hexokinase
  catalyzes the transfer of a phosphoryl group from
  ATP to glucose. Its Enzyme Commision number (EC
  number) is 2.7.1.1
• The first digit denotes the class name
  (transferase)
• The second digit denotes the subclass
  (phosphotransferase)
• The third digit denotes the acceptor atom
  (hydroxyl group)
• The fourth digit denotes the acceptor (D-glucose)

ATP D-glucose
ADP D-glucose 6-phosphate
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The Importance of Binding Energy

• On the left are examples of reaction coordinates
  of an uncatalyzed reaction, and one that is
  enzyme catalyzed.
• The active sites of enzymes tend to be more
  complementary to the transition states of their
  respective reactions than they are to the actual
  substrates.
• This results in lowering the energy of the
  enzymetransition state complex, meaning, a
  lowering of the activation energy.
• In order for catalysis to be effective, the
  energy barrier between ES and EXt must be less
  than S and Xt.
• Notice that the binding of substrate to enzyme
  lowers the free energy of the ES complex relative
  to substrate. If the energy is lowered too much,
  without a greater lowering of EXt, then catalysis
  would not take place.

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Acidity
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Amino Acid Reactivity

• The aliphatic amino acids (A, V, L, I) contain no
  polar or functional chemical groups, and
  therefore do not play a mechanistic role in
  enzyme catalysis.
• The amino acid glycine (G), is similar in that it
  also contains no polar or functional chemical
  groups. It plays no known role in enzyme
  catalysis that proceeds via polar mechanisms
  (those that involve carbocations or carbanions).
  Within the last 15 years, glycine has been shown
  to be an important amino acid in some enzyme
  reactions that proceed via carbon-centered
  unpaired electrons, such as pyruvate
  formate-lyase, and the anaerobic ribonucleotide
  reductase.
• Proline also plays a very limited role in the
  actual shuttling of electrons in enzyme
  catalysis. Peptide bonds involving proline
  residues are frequently substrates of enzymes
  that will catalyze a cis-trans isomerization of
  the bond.

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Serine and Threonine
Serine and threonine are both polar, and are
important in hydrogen bonding. Their reactivity,
especially outside of the confines of enzymatic
catalysis is somewhat diminished due to the
relatively high pKa value of their hydroxyl
fucntionality (ca. 16). They will react however
with acety chloride under acidic conditions.
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Lysine
Lysine will react with a number of electrophilic
reagents. Acetic anhydride is one well-known
reagent.
The iminium ion (not the imine) is very reactive
towards NaBH4 to form a stable teriary amine.
NaBH4, but not NaCNBH4, will reduce ketones to
carbonyls. NaCNBH4 will reduce iminium ions to
teriary amines.
The ability of lysine to react with carbonyl
compounds to form an imine or iminium (Schiffs
base) ion is a very common reaction in
biochemistry.
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Histidine
Diethyl pyrocarbonate is the reagent of choice
for modifying histidine residues. It also reacts
with lysine residues. Histidine modification is
reversible upon addition of hydroxylamine.
Lysine modification is irreversible.
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The Amino Acid Cysteine