Biochemistry

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Biochemistry
Chapter 13 Enzymes Chapter 14 Mechanisms of
enzyme action Chapter 15 Enzyme regulation
Chapter 17 Metabolism- An overview Chapter 18
Glycolysis Chapter 19 The tricarboxylic acid
cycle Chapter 20 Electron transport oxidative
phosphorylation Chapter 21 Photosynthesis http/
/www.aqua.ntou.edu.tw/chlin/
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Chapter 13

• Enzymes Kinetics and Specificity
• Biochemistry
• by
• Reginald Garrett and Charles Grisham

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• What are enzymes, and what do they do?

• Biological Catalysts
• Increase the velocity of chemical reactions

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• What are enzymes, and what do they do?
• Thousands of chemical reactions are proceeding
  very rapidly at any given instant within all
  living cells
• Virtually all of these reactions are mediated by
  enzymes--proteins (and occasionally RNA)
  specialized to catalyze metabolic reactions
• Most cells quickly oxidize glucose, producing
  carbon dioxide and water and releasing lots of
  energy
• C6H12O6 6 O2 ? 6 CO2 6 H2O 2870 kJ of
  energy
• It does not occur under just normal conditions
• In living systems, enzymes are used to accelerate
  and control the rates of vitally important
  biochemical reactions

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Figure 13.1Reaction profile showing the large
DG for glucose oxidation, free energy change of
-2,870 kJ/mol catalysts lower DG, thereby
accelerating rate.
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• Enzymes are the agents of metabolic function
• Enzymes form metabolic pathways by which
• Nutrient molecules are degraded
• Energy is released and converted into
  metabolically useful forms
• Precursors are generated and transformed to
  create the literally thousands of distinctive
  biomolecules
• Situated at key junctions of metabolic pathways
  are specialized regulatory enzymes capable of
  sensing the momentary metabolic needs the cell
  and adjusting their catalytic rates accordingly

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Figure 13.2The breakdown of glucose by
glycolysis provides a prime example of a
metabolic pathway. Ten enzymes mediate the
reactions of glycolysis. Enzyme 4, fructose 1,6,
biphosphate aldolase, catalyzes the C-C bond-
breaking reaction in this pathway.
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13.1 What Characteristic Features Define
Enzymes?

• Enzymes are remarkably versatile biochemical
  catalyst that have in common three distinctive
  features
• Catalytic power
• The ratio of the enzyme-catalyzed rate of a
  reaction to the uncatalyzed rate
• Specificity
• The selectivity of enzymes for their substrates
• Regulation
• The rate of metabolic reactions is appropriate to
  cellular requirements

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Catalytic power

• Enzymes can accelerate reactions as much as 1016
  over uncatalyzed rates!
• Urease is a good example
• Catalyzed rate 3x104/sec
• Uncatalyzed rate 3x10 -10/sec
• Ratio is 1x1014 (catalytic power)

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Specificity

• Enzymes selectively recognize proper substances
  over other molecules
• The substances upon which an enzyme acts are
  traditionally called substrates
• Enzymes produce products in very high yields -
  often much greater than 95

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Specificity

• The selective qualities of an enzyme are
  recognized as its specificity
• Specificity is controlled by structure of enzyme
• the unique fit of substrate with enzyme controls
  the selectivity for substrate and the product
  yield
• The specific site on the enzyme where substrate
  binds and catalysis occurs is called the active
  site

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Regulation

• Regulation of an enzyme activity is essential to
  the integration and regulation of metabolism
• Because most enzymes are proteins, we can
  anticipate that the functional attributes of
  enzymes are due to the remarkable versatility
  found in protein structure
• Enzyme regulation is achieved in a variety of
  ways, ranging from controls over the amount of
  enzyme protein produced by the cell to more
  rapid, reversible interactions of the enzyme with
  metabolic inhibitors and activators (chapter 15)

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Nomenclature

• Traditionally, enzymes often were named by adding
  the suffix -ase to the name of the substrate upon
  which they acted Urease for the urea-hydrolyzing
  enzyme or phosphatase for enzymes hydrolyzing
  phosphoryl groups from organic phosphate
  compounds
• Resemblance to their activity protease for the
  proteolytic enzyme
• Trypsin and pepsin

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Nomenclature

• International Union of Biochemistry and Molecular
  Biology (IUBMB)
• http//www.chem.qmw.ac.uk/iubmb/enzyme/
• Enzymes Commission number EC ...
• A series of four number severe to specify a
  particular enzyme
• First number is class (1-6)
• Second number is subclass
• Third number is sub-subclass
• Fourth number is individual entry

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• Classification of protein enzymes
• Oxidoreductases catalyze oxidation-reduction
  reactions
• Transferases catalyze transfer of functional
  groups from one molecule to another
• Hydrolases catalyze hydrolysis reactions
• Lyases catalyze removal of a group from or
  addition of a group to a double bond, or other
  cleavages involving electron rearrangement
• Isomerases catalyze intramolecular rearrangement
  (isomerization reactions)
• Ligases catalyze reactions in which two molecules
  are joined (formation of bonds)

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• For example, ATPD-glucose-6-phosphotransferase
  (glucokinase) is listed as EC 2.7.1.2.
• ATP D-glucose ? ADP D-glucose-6-phospha
  te
• A phosphate group is transferred from ATP to
  C-6-OH group of glucose, so the enzyme is a
  transferase (class 2)
• Transferring phosphorus-containing groups is
  subclass 7
• An alcohol group (-OH) as an acceptor is
  sub-subclass 1
• Entry 2

EC 2.7.1.1 hexokinaseEC 2.7.1.2 glucokinaseEC
2.7.1.3 ketohexokinaseEC 2.7.1.4 fructokinaseEC
2.7.1.5 rhamnulokinaseEC 2.7.1.6
galactokinaseEC 2.7.1.7 mannokinase EC 2.7.1.8
glucosamine kinase . .. . EC 2.7.1.156
adenosylcobinamide kinase
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• Many enzymes require non-protein components
  called coenzymes or cofactors to aid in catalysis
• Coenzymes many essential vitamins are
  constituents of coenzyme
• Cofactors metal ions
• metalloenzymes
• Holoenzyme apoenzyme (protien) prosthetic
  group

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Other Aspects of Enzymes

• Mechanisms - to be covered in Chapter 14
• Regulation - to be covered in Chapter 15
• Coenzymes - to be covered in Chapter 17

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13.2 Can the Rate of an Enzyme-Catalyzed
Reaction Be Defined in a Mathematical Way?

• Kinetics is concerned with the rates of chemical
  reactions
• Enzyme kinetics addresses the biological roles of
  enzymatic catalyst and how they accomplish their
  remarkable feats
• In enzyme kinetics, we seek to determine the
  maximum reaction velocity that the enzyme can
  attain and its binding affinities for substrates
  and inhibitors
• These information can be exploited to control and
  manipulate the course of metabolic events

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Chemical kinetics

• A ? P
• (A ? I ? J ? P)
• rate or velocity (v)
• v dP / dt or v -dA / dt
• The mathematical relationship between reaction
  rate and concentration of reactant(s) is the rate
  law
• v -dA / dt k A
• k is the proportional constant or rate constant
  (the unit of k is sec-1)

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Chemical kinetics

• v -dA / dt k A
• v is first-order with respect to A
• The order of this reaction is a first-order
  reaction
• molecularity of a reaction
• The molecularity of this reaction equal 1
  (unimolecular reaction)

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Figure 13.4Plot of the course of a first-order
reaction. The half-time, t1/2, is the time for
one-half of the starting amount of A to
disappear.
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Chemical kinetics

• A B ? P Q
• The molecularity of this reaction equal 2
  (bimolecular reaction)
• The rate or velocity (v)
• v -dA / dt -dB / dt dP / dt
  dQ / dt
• The rate law is
• v k A B
• The order of this reaction is a second-order
  reaction
• The rate constant k has the unit of M-1 sec-1)

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The Transition State

• Reaction coordinate a generalized measure of the
  progress of the reaction
• Free energy (G)
• Standard state free energy (25?, 1 atm, 1 M/each)
• Transition state
• The transition state represents an intermediate
  molecular state having a high free energy in the
  reaction.
• Activation energy
• Barriers to chemical reactions occur because a
  reactant molecule must pass through a high-energy
  transition state to form products.
• This free energy barrier is called the activation
  energy.

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Decreasing ?G increase reaction rate

• Two general ways may accelerate rates of chemical
  reactions
• Raise the temperature
• The reaction rate are doubled by a 10?
• Add catalysts
• True catalysts participate in the reaction, but
  are unchanged by it. Therefore, they can continue
  to catalyze subsequent reactions.
• Catalysts change the rates of reactions, but do
  not affect the equilibrium of a reaction.

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(a) Raising the temperate
(b) Adding a catalyst
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• Most biological catalysts are proteins called
  enzymes (E).
• The substance acted on by an enzyme is called a
  substrate (S).
• Enzymes accelerate reactions by lowering the free
  energy of activation
• Enzymes do this by binding the transition state
  of the reaction better than the substrate
• The mechanism of enzyme action in Chapter 14

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13.3 What Equations Define the Kinetics of
Enzyme-Catalyzed Reactions?

• The Michaelis-Menten Equation
• The Lineweaver-Burk double-reciprocal plot
• Hanes-Woolf plot

Vmax S
v
Km S
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• Figure 13.7 Substrate saturation curve for an
  enzyme-catalyzed reaction. The amount of enzyme
  is constant, and the velocity of the reaction is
  determined at various substrate concentrations.
  The reaction rate, v, as a function of S is
  described by a rectangular hyperbola. At very
  high S, v Vmax. The H2O molecule provides a
  rough guide to scale. The substrate is bound at
  the active site of the enzyme.

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The Michaelis-Menten Equation

• Louis Michaelis and Maud Menten's theory
• It assumes the formation of an enzyme-substrate
  complex (ES)
• E S ES
• At equilibrium
• k-1 ES k1 E S
• And
• Ks

k1
k-1
E S
k-1
ES
k1
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The Michaelis-Menten Equation

• E S ES E
  P
• The steady-state assumption
• ES is formed rapidly from E S as it disappears
  by dissociation to generate E S and reaction to
  form E P
• dES
• dt
• That is formation of ES breakdown of ES
• k1 E S k-1ES k2ES

k1
k2
k-1
0
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Figure 13.8Time course for the consumption of
substrate, the formation of product, and the
establishment of a steady-state level of the
enzyme-substrate ES complex for a typical
enzyme obeying the Michaelis-Menten,
Briggs-Haldane models for enzyme kinetics. The
early stage of the time course is shown in
greater magnification in the bottom graph.
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The Michaelis-Menten Equation

• k1 E S k-1ES k2ES
  (k-1 k2) ES
• ES ( ) E S
• Km
• Km is Michaelis constant
• Km ES E S

k1
k-1 k2
k-1 k2
k1
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The Michaelis-Menten Equation

• Km ES E S
• Total enzyme, ET E ES
• E ET ES
• Km ES (ET ES) S ET
  S ES S
• Km ES ES S ET S
• (Km S) ES ET S
• ES

ET S
Km S
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The Michaelis-Menten Equation

• ES
• The rate of product formation is
• v k2 ES
• v
• Vmax k2 ET v

ET S
Km S
k2 ET S
Km S
Vmax S
Km S
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Understanding Km

• The Michaelis constant Km measures the substrate
  concentration at which the reaction rate is
  Vmax/2.
• associated with the affinity of enzyme for
  substrate
• Small Km means tight binding high Km means weak
  binding

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v When v Vmax / 2 Vmax Vmax
S 2 Km S Km S 2 S S
Km
Vmax S
Km S

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Understanding Vmax

• The theoretical maximal velocity
• Vmax is a constant
• Vmax is the theoretical maximal rate of the
  reaction - but it is NEVER achieved in reality
• To reach Vmax would require that ALL enzyme
  molecules are tightly bound with substrate
• Vmax is asymptotically approached as substrate is
  increased

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The dual nature of the Michaelis-Menten equation

• Combination of 0-order and 1st-order kinetics
• When S is low (s ltlt Km), the equation for rate
  is 1st order in S
• When S is high (s gtgtKm), the equation for rate
  is 0-order in S
• The Michaelis-Menten equation describes a
  rectangular hyperbolic dependence of v on S
• The actual estimation of Vmax and consequently Km
  is only approximate from each graph

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The turnover number

• A measure of catalytic activity
• kcat, the turnover number, is the number of
  substrate molecules converted to product per
  enzyme molecule per unit of time, when E is
  saturated with substrate.
• kcat is a measure of its maximal catalytic
  activity
• If the M-M model fits, k2 kcat Vmax/Et
• Values of kcat range from less than 1/sec to many
  millions per sec (Table 13.4)

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The catalytic efficiency

• Name for kcat/KmAn estimate of "how perfect" the
  enzyme is
• kcat/Km is an apparent second-order rate constant
  
• v (kcat/Km) E S
• kcat/Km provides an index of the catalytic
  efficiency of an enzyme
• kcat/Km k1 k2 / (k-1 k2)
• The upper limit for kcat/Km is the diffusion
  limit - the rate at which E and S diffuse
  together

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Linear Plots of the Michaelis-Menten Equation

• Lineweaver-Burk plot
• Hanes-Woolf plot
• Smaller and more consistent errors across the plot

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V 1 Km S V Vmax S
Vmax S
Km S

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13.4 What Can Be Learned from the Inhibition of
Enzyme Activity?

• Enzymes may be inhibited reversibly or
  irreversibly
• Reversible inhibitors may bind at the active site
  (competitive) or at some other site
  (noncompetitive)
• Enzymes may also be inhibited in an irreversible
  manner
• Penicillin is an irreversible suicide inhibitor

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Competitive inhibition
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k1
kcat
E S ES E
P I EI
k-1
k3
k-3
A competitive inhibitor competes with substrate
for the binding site. It changes the apparent km.
kcat Et S
kcat Et S
VmaxS
V

app
app
km (1 I/ KI) S
km S
km S
I
app
km km (1 )
KI k-3 / k3
KI
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Figure 13.14Structures of succinate, the
substrate of succinate dehydrogenase (SDH), and
malonate, the competitive inhibitor. Fumarate
(the product of SDH action on succinate) is also
shown.
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Noncompetitive inhibition
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k1
kcat
E S ES E P
I
I EI S EIS
k-1
k3
k-3
k-3
k3
k1
k-1
app
app
kcat Et S
Vmax S
kcat (1 I/ KI) Et S
V

km S
km S
km S
I
app
Vmax Vmax (1 )
KI
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KI KI
Figure 13.15Lineweaver-Burk plot of pure
noncompetitive inhibition. Note that I does not
alter Km but that it decreases Vmax. In the
presence of I, the y-intercept is equal to
(1/Vmax)(1 I/KI).
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KI KI
Figure 13.16Lineweaver-Burk plot of mixed
noncompetitive inhibition. Note that both
intercepts and the slope change in the presence
of I. (a) When KI is less than KI' (b) when KI
is greater than KI'.
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Uncompetitive inhibition
k1
kcat
E S ES E P

I EIS
k-1
k-3
k3
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Figure 13.17Lineweaver-Burk plot of pure
uncompetitive inhibition. Note that I does not
alter Km but that it decreases Vmax. In the
presence of I, the y-intercept is equal to
(1/Vmax)(1 I/KI).
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Irreversible inhibition

• Irreversible inhibition occurs when substances
  combine covalently with enzymes so as to
  inactivate them irreversibly.
• Suicide substrates are inhibitory substrate
  analogs designed, via normal catalytic actions of
  the enzyme, a very reactive group is generated.
  This reactive group then forms a covalent bond
  with a nearby functional group within the active
  site of the enzyme, thereby causing irreversible
  inhibition
• Almost all irreversible enzyme inhibitors are
  toxic substances, either natural or synthetic.
  Such as penicillin

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Figure 13.18 Penicillin is an irreversible
inhibitor of the enzyme glycoprotein peptidase,
which catalyzes an essential step in bacterial
cell wall synthesis.
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13.5 - What Is the Kinetic Behavior of Enzymes
Catalyzing Bimolecular Reactions?

• Enzymes often use two (or more) substrates
• Bisubstrate reactions
• A B P Q
• Reactions may be sequential or single-displacement
  reactions (both A and B are bound to the enzyme)
• E A B ? AEB ? PEQ ? E P Q
• And they can be random or ordered
• Ping-pong or double-displacement reactions

enzyme
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Figure 13.19 Single-displacement bisubstrate
mechanism.
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The conversion of AEB to PEQ is the rate-limiting
step in random, single-displacement reactions
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Figure 13.20 Random, single-displacement
bisubstrate mechanisms where A does not affect B
binding, and vice versa
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In an ordered, single-displacement reaction
Similar to 1st-order reaction
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double-displacement (ping-pong) reactions
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Glutamateaspartate aminotransferase
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13.6 How Can Enzymes Be So Specific?

• Lock and key hypothesis was the first
  explanation for specificity
• Induced fit provides a more accurate
  description
• Induced fit favors formation of the
  transition-state intermediate

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Figure 13.24 A drawing, roughly to scale, of
H2O, glycerol, glucose, and an idealized
hexokinase molecule
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13.7 Are All Enzymes Proteins?

• Relatively new discoveries
• RNA molecules that are catalytic have been termed
  Ribozymes
• Examples RNase P and peptidyl transferase
• The ribosome is a ribozyme
• Antibody molecules can have catalytic activity
  (called Abzymes) - antibodies raised to bind the
  transition state of a reaction of interest

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Figure 13.25RNA splicing in Tetrahymena rRNA
maturation (a) the guanosine-mediated reaction
involved in the autocatalytic excision of the
Tetrahymena rRNA intron, and (b) the overall
splicing process. The cyclized intron is formed
via nucleophilic attack of the 3'-OH on the
phosphodiester bond that is 15 nucleotides from
the 5'-GA end of the spliced-out intron.
Cyclization frees a linear 15-mer with a 5'-GA
end.
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Figure 13.26 (a) The 50S subunit from H.
marismortui. (b) The aminoacyl-tRNA (yellow) and
the peptidyl-tRNA (orange) in the peptidyl
transferase active site.
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The cyclic phosphonate ester analog of the cyclic
transition state
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13.8 Is It Possible to Design An Enzyme to
Catalyze Any Desired Reaction?

• A known enzyme can be engineered by in vitro
  mutagenesis, replacing active site residues with
  new ones that might catalyze a desired reaction
• Another approach attempts to design a totally new
  protein with the desired structure and activity
• This latter approach often begins with studies
  in silico i.e., computer modeling
• Protein folding and stability issues make this
  approach more difficult
• And the cellular environment may provide
  complications not apparent in the computer
  modeling

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limited
rapidly
Figure 13.29 cis-1,2-Dichloroethylene (DCE) is
an industrial solvent that poses hazards to human
health. Site-directed mutations (F108L, I129L,
and C248I) have enabled the conversion of a
bacterial epoxide hydrolase to catalyze the
chlorinated epoxide hydrolase reaction.