Chapter 6: Outline1

1
Chapter 6 Outline-1

• Properties of Enzymes
• Classification of Enzymes
• Enzyme Kinetics
• Michaelis-Menten Kinetics
• Lineweaver-Burke Plots
• Enzyme Inhibition
• Catalysis
• Catalytic Mechanisms
• Cofactors

2
Chapter 6 Outline-2

• Catalysis cont.
• Temperature and pH
• Detailed Mechanisms
• Genetic Control
• Enzyme Regulation
• Covalent Modification
• Allosteric Regulation
• Compartmentation

3
Introduction

• The proteins which serve as enzymes, Mother
  Natures catalysts, are globular in nature.
  Because of their complex molecular structures,
  they often have exquisite specificity for their
  substrate molecule and can speed up a reaction by
  a factor of millions relative to an uncatalyzed
  reaction. This presentation will describe how
  enzymes function.

4
6.1 Properties of Enzymes

• A catalyst enhances the rate of reaction but is
  not permanently altered.
• Catalysts work by decreasing the activation
  energy for a reaction.
• The structure of the active site of the enzyme
  (shape and charge distribution) is used to
  optimally orient the substrate for reaction.
• The energy of the enzyme-substrate complex is
  then closer to the TS.

5
Activation Energy, Eact

• An enzyme speeds a reaction by lowering the
  activation energy. It does this by changing the
  reaction pathway.

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Activation Energy-2

• An enzyme lowers the activation energy but it
  does not change the standard free energy change
  (DG) for the reaction nor the Keq.
• A catalyst cannot make an endergonic reaction
  exergonic or vice versa.
• Most enzymes are temperature/pH sensitive and
  will not work outside their normal temperature/pH
  range because the enzyme is denatured.

7
Enzymes Models

• In the lock-and-key model, the enzyme is assumed
  to be the lock and the substrate the key. The
  two are made to fit exactly. This model fails to
  take into account the fact that proteins can and
  do change their conformations to accommodate a
  substrate molecule.

8
Enzymes Models-2

• The induced-fit model of enzyme action assumes
  that the enzyme conformation changes to
  accommodate the substrate molecule. Eg.

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6.2 Classification of Enzymes

• The International Union of Biochemistry (IUB)
  classifies and names enzymes according to the
  type of chemical reaction it catalyzes.
• Enzymes are assigned a four-number class and a
  systematic two-part name.
• A shorter recommended name is also suggested.
• Alcohol dehydrogenase is
• alcoholNAD oxidoreductase
• (E.C. 1.1.1.1)

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Enzyme Classes

• 1. Oxidoreductases catalyze redox reactions. Eg.
  Reductases or peroxidases
• 2. Transferases transfer a group from one
  molecule to another. Eg. Transaminases,
  transcarboxylases
• 3. Hydrolases cleave bonds by adding water. Eg.
  Phosphatases or peptidases

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Enzyme Classes-2

• 4. Lyases catalyze removal of groups to form
  double bonds or the reverse. Eg. decarboxylases
  or synthases
• 5. Isomerases catalyze intramolecular
  rearrangements. Eg. epimerases or mutases
• 6. Ligases bond two molecules together. Many are
  called synthetases. Eg. carboxylases

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Enzyme Classes-3 Examples
13
Enzyme Classes-3 Examples
14
6.3 Enzyme Kinetics

• Kinetics is the field of chemistry that studies
  the rate and mechanism of a reaction.
• Rates are usually measured in terms of how many
  moles of reactant or product are changed per time
  period.
• A mechanism is a detailed step-by-step
  description of how a reaction occurs at the
  molecular level.

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The Rate Equation-1
A ? P Init. Rate vo - DA or DP

Dt
Dt D change in, A
molarity and t is time. Disappearance of
reactants is negative so the quantity has a
negative sign to make all rates positive. First
order Rate DA kA1
Dt
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The Rate Equation-2

• Rate k Ax The rate equals the experimentally
  determined rate constant, k, times the
  concentrations of A to some experimentally
  determined power, x. Values for x are frequently
  0, 1 or 2.
• ALL RATE EQUATIONS ARE DETERMINED EXPERIMENTALLY!!

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Enzyme Rxns Type 1

• Chymotrypsin cleaves proteins at the COOH end of
  aromatic side chain AAs.
• At low substrate concentrations, the reaction is
  first order in substrate.
• As the concentration of substrate increases, the
  order changes and approaches zero.
• A graph of velocity vs substrate conc. is
  hyperbolic. Nonallosteric (See graph, 22)

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Enzyme Rxns Type 1

• Chymo-trypsin Hyperbolic plot

Conc. At ½ max velocity
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Enzyme Rxns Type 2

• Aspartate transcarbamoylase (ATCase) catalyzes
  the reacton between aspartate and carbamoyl
  phosphate.
• This reaction leads ultimately to the synthesis
  of nucleobases needed for DNA and RNA synthesis.
• Velocity as a function of aspartate concentration
  gives a sigmoidal plot. Allosteric (See 24)

20
Enzyme Rxns Type 2
ATCase Sigmoidal plot
21
Michaelis-Menten Kinetics-1

• M-M kinetics explains the behavior of
  nonallosteric enzymes. It assumes an
  enzyme-substrate complex is formed.

At low substrate concentrations, the reaction is
first order with respect to substrate. At high
substrate concentrations, the enzyme is saturated
with substrate. The order is zero and a Vmax
occurs.
At low substrate concentrations, the reaction is
first order with respect to substrate. At high
substrate concentrations, the enzyme is saturated
with substrate. The order is zero and a Vmax
occurs.
22
Michaelis-Menten Kinetics-3

• Michaelis and Menten also derived what is now
  known as the Michaelis-Menten equation.

Vmax max velocity The lower the Km, the greater
the affinity for complex formation.
Vmax max velocity The lower the Km, the greater
the affinity for complex formation.
Vmax max velocity The lower the Km, the greater
the affinity for complex formation.
An enzymess kinetic properties can be used to
determine its catalytic efficiency. (Next slide.)
23
Michaelis-Menten Kinetics-5

• Turnover numbers for some enzymes follow. They
  vary greatly!!
• Enzyme kcat (s-1)
• Catalase 10,000,000
• Chymotrypsin 190
• Lysozyme 0.5
• Note catalyse turns over 10 milliion molecules
  of substrate per sec!!

24
Michaelis-Menten Kinetics-6

• Substrate concentration at ½ Vmax is termed the
  KM (Michaelis constant) for the reaction.
  Nonallosteric

KM is difficult to measure by this method as
Vmax must be estimated. A linear plot gives
better results.
25
Lineweaver-Burk Plot

• A Lineweaver-Burk plot for nonallosteric enzymes
  gives a straight line and better data to
  determine KM.

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Lineweaver-Burk Plot

• A L-B plot of 1/V vs 1/S is shown below

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Enzyme Inhibition

• Inhibitors interfere with enzyme action.
• They may be reversible or irreversible.
• The three kinds of reversible inhibitors are
  competitive, uncompetitive, and noncompetitive .
• A competitive inhibitor looks structurally like
  the substrate and binds to the enzyme at the
  active site.
• An uncompetative inhibitor binds only to the
  enzyme-substrate complex.
• A noncompetitive inhibitor does not look like
  substrate and binds at a site other than the
  active site.

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Competitive Inhibitor-2

• Since a competitive inhibitor competes with
  substrate for the active site, its influence can
  be negated with large concentrations of
  substrate. Thus the Vmax remains constant.
• Since the velocity is slower compared to normal
  substrate concentrations, the slope of the L-B
  line increases and the KM increases.
• The effect of a competitive inhibitor on a L-B
  plot is shown on slide 42.

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Uncompetitive Inhibitor-2

• Since an uncompetitive inhibitor binds only to
  the enzyme-substrate complex, adding more
  substrate will increase the rate but not to the
  original values without inhibitor.
• Commonly observed when the enzyme binds to more
  than one substrate.

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Noncompetitive Inhibitor-2

• For a noncompetitive inhibitor, the velocity of
  the reaction is slowed at all substrate
  concentrations. Thus the Vmax is permanently
  lowered.
• The slope of the L-B line increases but KM stays
  constant.
• The effect of a noncompetitive inhibitor on a L-B
  plot is shown on slide 42.

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Kinetics Inhibition
32
Chapter 6 Outline-1

• Catalysis (We are here.)
• Catalytic Mechanisms
• Cofactors
• Temperature and pH
• Detailed Mechanisms
• Genetic Control
• Enzyme Regulation
• Covalent Modification
• Allosteric Regulation
• Compartmentation

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Introduction

• This presentation covers catalytic mechanisms,
  cofactors, and enzyme regulation.
• Remember, allosteric enzymes show cooperative
  binding. As the first substrate binds it
  influences subsequent binding.

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6.4 Catalysis

• How does the substrate interact with the enzyme?
• Proximity and Strain Effects
• Substrate closely aproaches the catalytic site
  with proper orientation. Enzyme conformation
  probably changes to give a strained E-S complex.
• Electrostatic Effects
• A hydrophobic pocket in the enzyme lowers the
  surrounding dielectric constant allowing
  electrostatic interaction between E and S.

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Catalytic Mechanisms-2

• Acid-Base catalysis
• Enzyme side chains act as proton donors and
  acceptors.

• Covalent Catalysis
• A nucleophilic side chain forms an unstable
  covalent bond to the substrate.

36
Cofactors Metals

• Transition metals are often involved in
  catalysis, eg. Fe3, Cu2, Co 2, (Zn 2 ).
• They are useful because they
• Have a high positive charge density.
• Act as Lewis acids (accept e pairs).
• Can mediate redox reactions. (Fe3/2)
• Help polarize water molelcules.

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Cofactors Coenzymes

• Coenzymes are organic molecules often derived
  from vitamins.
• Vitamin Coenzyme Process
• Thiamine(B1) TTP decarboxylation
• Niacin NAD(P) redox
• Riboflavin(B6) Pyridoxal P amino group transfer
• Folic acid THF one-carbon transfer
• Vit A retinal vision, growth

38
Cofactors Coenzymes-2

• Nicotinic acid (niacin) is involved in redox
  reactions.

39
Cofactors Coenzymes-3

• The nicotinamide part of NAD accepts a hydride
  (H plus two electrons) from the alcohol to be
  oxidized. The alcohol loses a proton to the
  solvent.

Ox form
Red form
40
Cofactors Coenzymes-4

• Flavin coenzymes also serve in redox reactions

41
Cofactors Coenzymes-5

• The flavin coenzymes accept electrons in the
  flavin ring system.

42
Temperature and pH

• An enzyme has an optimum temperature that is
  usually close to the temperature at which it
  normally works, ie. 37 oC for humans. Excessive
  heat can denature a protein.

43
Temperature and pH-2

• Enzymes work best at the correct physiological
  pH. Extreme pH changes will denature the enzyme.
  Pepsin (stomach) and chymotrypsin (small
  intestine) have different optimum pHs.

Chymo- trypsin
pepsin
44
Mechanism for Chymtrypsin

• Chymotrypsin (CT) catalyzes the hydrolysis of
  peptide bonds next to aromatic side chains.
• The active site on CT involves the serine 195
  residue. (CT is a serine protease.)
• This was determined by labeling the serine with
  diisopropylphosphofluoridate

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Mechanism for Chymtrypsin-2

• The active site on CT also involves the
  histidine 57 residue.
• Ser 195 and His 57 are close together in the
  active site of the enzyme.
• The histidine acts as a general base catalyst,
  converting the serine to its anion and making it
  a better nucleophile for attack at the carbonyl
  carbon to be hydroylzed.

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Mechanism for Chymtrypsin-3
47
Mechanism for Chymtrypsin-4
First tetrahedral intermediate
The C-N bond cleaves. Original serine proton
transfers to nitrogen.
48
Mechanism for Chymtrypsin-5
49
Mechanism for Chymtrypsin-6
His-57 again serves as a general base catalyst.
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Mechanism for Chymtrypsin-7
And decomposes to give product C-term Phe
protein
51
Mechanism for Chymtrypsin-8
C-term protein
52
Regulating Enzymes

• Some methods that organisms use to regulate
  enzyme activity are
• Genetic control
• Covalent modification
• Allosteric regulation
• Compartmentation

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1. Genetic Control

• An example of enzyme induction is when E. coli is
  induced to use lactose as an energy source in the
  absence of glucose. The enzyme for lactose use
  is turned on by genetic control.
• In enzyme repression, the product of a
  biochemical pathway inhibits the functioning of a
  key enzyme of a previous step in the pathway.

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2. Covalent Modification

• Phosphorylation/dephosphorylation is a common way
  to control enzyme activity. Glycogen
  phosphorylase is a good example of an enzyme
  using this mechanism.
• Methylation and acetylation are two other
  examples of covalent modification.
• Conversion of zymogens (preenzymes) to active
  enzymes is another example.

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3. Allosteric Regulation

• Pacemaker (regulatory) enzymes usually catalyze
  the committed step in a series of biochemical
  reactions or a step where branching to two paths
  can occur.
• Often these enzymes are allosteric enzymes (See
  Proteins II) and usually they are composed of
  several protomers.

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3. Allosteric Regulation-2

• Allosteric ligands (effectors) can be positive or
  negative.
• Eg CTP is an inhibitor of ATCase activity or a
  negative effector
• ATP is an activator of ATCase or a positive
  effector.
• The graph on Slide 28 shows the effect of
  positive and negative effectors on an allosteric
  enzyme.

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3. Allosteric Regulation-3
activator
inhibitor
58
Allosteric Models-1

• The concerted model assumes the enzyme has two
  states T(taut) and R (relaxed). Substrates and
  activators bind easily to the R form while
  inhibitors bind more easily to the T form.
• The first effector to bind changes the
  conformation of all the protomers simultaneously
  thereby greatly promoting activation or
  inhibition.

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Allosteric Models-2

• The sequential model is needed to explain
  negative cooperativity, a situation in which the
  binding of the first ligand reduces the affinity
  for similar ligands.
• In this model, the first ligand is assumed to
  induce conformational changes that are
  transmitted sequentially to other protomers in
  the enzyme.
• Neither model above fully explains all allosteric
  enzyme activity.

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Allosteric Models-3

• The pictures below attempt to show the difference
  between the concerted and the sequential models
  of allosteric enzyme behavior.

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4. Compartmentation

• Eukaryotic cells are divided into organelles
  which often allows for separation of opposing
  processes.
• Eg. Fatty acid oxidation occurs in the
  mitochondria while synthesis occurs in the
  cytosol.
• Organelles also allow for concentration of
  specific reagents. Eg. Lysosomes require a low
  pH (5) and their membrane keeps the high H
  inside.

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Medical Apps Diagnosis

• To confirm a heart attack and monitor the
  treatment, doctors use creatine kinase (CK) and
  lactate dehydrogenase (LDH) which are found in
  blood serum.
• Both enzymes exist in multiple forms called
  isozymes which have slightly different AA
  sequences.
• The forms are separable by electrophoresis which
  gives characteristic patterns after an infarction.

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Medical Apps Diagnosis-2

• Dimeric CK has two types of protomers, muscle (M)
  and brain (B).
• Heart muscle has CK2 (MB) and CK3 (MM). Only CK2
  is found exclusively in heart muscle. (See
  graph.)

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Medical Apps Diagnosis-3

• LDH is a tetramer composed of two protomers,
  heart (H) and muscle (M). Of the five LDH
  isozymes, LDH1 (H4) and LDH2 (H3M) are found
  only in heart muscle and red blood cells.
• Again, electrophoresis patterns can be used to
  diagnose an infarct. The next slide shows normal
  and abnormal patterns for LDH1-5.

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Medical Apps Diagnosis-4
Normal LDH electrophoresis pattern
LDH electrophoresis pattern after infarct
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Medical Apps Therapy

• Streptokinase and human tissue plasminogen
  activator (tPA) are both used to treat heart
  attack because they dissolve blood clots
• Asparaginase does not occur in human blood. Some
  cancer cells (eg some adult leukemias) cannot
  synthasize asparagine. Infusing the enzyme can
  cause cancer cell death due to lack of
  asparagine. Serious side affects can occur.