Voet:

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Voet Suggested Problems Ch 10 1, 3, 4, 5,
6, 8, 10, 11, 12, Segel Chapter 1 53,
55 2 1, 2, 13, 30,
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Suggested problems chapter 13 3, 4, 5, 7,
8 Chapter 14 3, 4, 5, 6, 12
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Table 13-3 Enzyme Classification According to
Reaction Type.
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Figure 13-1 An enzymesubstrate complex
illustrating both the geometric and the physical
complementarity between enzymes and substrates.
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Figure 13-3 Prochiral differentiation.
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Trypsin inhibitor (VVP Fig 11-28)
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Common features of enzyme active sites
1. The active site is a 3-dimensional cleft
formed from amino acids at distant sites in the
sequence.
2. The active site accounts for a relatively
small part of the total volume of the protein.
3. Substrates are generally bound to enzymes by
non-covalent interactions.
4. The specificity of S binding depends on the
arrangements of atoms in the active site.
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Number Classification Biochemical Properties
1. Oxidoreductases Act on many
chemical groupings to add or remove hydrogen
atoms. 2. Transferases Transfer functional
groups between donor and acceptor
molecules. Kinases are specialized transferases
that regulate metabolism by transferring
phosphate from ATP to other molecules. 3. Hydr
olases Add water across a bond, hydrolyzing
it. 4. Lyases Add water, ammonia or carbon
dioxide across double bonds, or remove these
elements to produce double bonds.
5. Isomerases Carry out many kinds of
isomerization L to D isomerizations, mutase
reactions (shifts of chemical groups) and
others. 6. Ligases Catalyze reactions in which
two chemical groups are joined (or ligated)
using ATP.
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1.Addition or removal of water
a.Hydrolases - these include esterases,
carbohydrases, nucleases, deaminases, amidases,
and proteases b.Hydrases such as
fumarase, enolase, aconitase and carbonic
anhydrase 2.Transfer of electrons
a.Oxidases b.Dehydrogenases
3.Transfer of a radical
a.Transglycosidases - of monosaccharides
b.Transphosphorylases and phosphomutases - of a
phosphate group c.Transaminases - of
amino group d.Transmethylases - of a
methyl group e.Transacetylases - of an
acetyl group 4.Splitting or forming a C-C bond
a.Desmolases 5.Changing geometry or
structure of a molecule a.Isomerases
6.Joining two molecules through hydrolysis of
pyrophosphate bond in ATP or other triphosphate
a.Ligases
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Oxidoreductases--catalyze redox reactions
Usually require a coenzyme
Ethanol NAD ? Acetaldehyde NADH H
Enzymes receive common names reflecting their
function, either in the forward or reverse
direction.
The enzyme for this reaction is called
Alcohol Dehydrogenase
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Transferases-transfer functional groups
Kinases transfer phosphates from ATP (or GTP)
E.g Hexokinase Glucose ATP ?glc-6-P ADP
Hydrolases catalyze hydrolytic cleavages
Proteases are hydrolases
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Lyases catalyze group elimination to form double
bonds
e.g. Enolase (glycolysis)
2-Phosphoglycerate ? H2O phosphoenolpyruvate
Isomerases--duh, interconvert isomers
e.g. phosphoglucose isomerase
Glucose-6-phosphate ? Fructose-6-phosphate
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Ligases--join to substrates together at the
expense of ATP
e.g. DNA Ligase
Joins Okazaki fragments during DNA replication
Some bacterial ligases substitute NAD as the
energy source.
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Coenzymes

• Enzymes often require the participation of other
  small molecules to carry out a particular
  reaction.
• These small molecules, called coenzymes, are
  metabolic derivatives of vitamins.
• Vitamins are nutrients required in small amounts
  by organisms. Vitamin deficiencies usually
  present as metabolic disorders, e.g. scurvy

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Table 13-1 The Common Coenzymes.
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Table 13-2 Vitamins That Are Coenzyme Precursors.
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Figure 13-2 The structures and reaction of
nicotinamide-adenine dinucleotide (NAD) and
nicotinamide adenine dinucleotide phosphate
(NADP).
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Figure 13-4 Structures of nicotinamide and
nicotinic acid.
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Enzyme Activities Are Regulated at Various Levels

• Transcription
• Processing
• Translation
• Post-translational modification
• Transient modification (e.g. phosphorylation)
• Allosteric Effectors

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Figure 13-5 The rate of the reaction catalyzed by
ATCase as a function of aspartate concentration.
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Figure 13-6 Schematic representation of the
pyrimidine biosynthesis pathway.
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Figure 13-7a X-Ray structure of ATCase. (a)
(left) T-state ATCase along the proteins
molecular threefold axis of symmetry (right)
R-state ATCase along the proteins molecular
threefold axis of symmetry.
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Figure 13-7b X-Ray structure of ATCase. (b)
(left) T-state ATCase along the proteins
molecular twofold axis of symmetry (right)
R-state ATCase along the proteins molecular
twofold axis of symmetry.
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Figure 13-8 Comparison of the polypeptide
backbones of the ATCase catalytic subunit in the
T state (orange) and the R state (blue).
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Figure 13-9 Schematic diagram indicating the
tertiary and quaternary conformational changes in
two vertically interacting catalytic ATCase
subunits.
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Styer p.277
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Stryer p. 278
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Figure 18-12 A bicyclic enzyme cascade.
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Figure 18-13 Control of glycogen metabolism
in muscle.
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Stryer Fig. 10.37
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Ch 14 Kinetics!
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Stryer Fig. 8.11
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Stryer Fig. 8.12 Determining initial velocity
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What other factors can affect enzyme activity?
Is the product stable or transformed into
something else?
E
pH
Temperature
Salt concentration
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After the first few milliseconds of a reaction,
a steady state is attained.
Stryer Fig. 8.13
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Figure 14-1 Plot of lnA versus time for a
first-order reaction.
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Figure 14-4b Transition state diagrams. (b) For a
spontaneous reaction, that is, one in which the
free energy decreases.
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Figure 14-5 Transition state diagram for the
two-step overall reaction A I P.
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Figure 14-6 The effect of a catalyst on the
transition state diagram of a reaction.
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Figure 14-7 Progress curves for the components of
a simple MichaelisMenten reaction.
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Figure 14-8 Plot of the initial velocity vo of a
simple MichaelisMenten reaction versus the
substrate concentration S.
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k2
k1
E S ?ES?EP
k-1
(k-2 is negligible until products start to build
up)
Steady state conditions--ES remains relatively
constant over the course of the rxn until S
starts runing out.
Vo k2ES
k1ES k-1ES k2ES (k-1 k2)ES
Define a new constant ES/ES (k-1 k2)/
k1 KM
KmES SE
KmES ETS-ESS
E ET -ES
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ES(KM S) ETS
(ETS
And ES v/k2
ES ------------------- KM S
k2ETS
Define Vmaxk2ET
v ------------------ KM S
vmaxS
Michaelis-Menton equation
v ----------------- KM S
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Figure 14-1 Plot of lnA versus time for a
first-order reaction.
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Figure 14-8 Plot of the initial velocity vo of a
simple MichaelisMenten reaction versus the
substrate concentration S.
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An enzyme obeys Michaelis-Menten kinetics
with Vmax 1.8 umol ml-1 s-1 at an enzyme
concentration of 15 umol ml-1. Calculate kcat
and KM for the enzyme. Is the value you obtain
for KM what you would expect given your data?
Why or why not? S uM vo (umol ml-1
s-1) 1600 1.39 800 1.13 400 0.83 200
0.54 100 0.32
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An enzyme obeys Michaelis-Menten kinetics
with Vmax 1.8 umol ml-1 s-1 at an enzyme
concentration of 15 umol ml-1. Calculate kcat
and KM for the enzyme. Is the value you obtain
for KM what you would expect given your data?
Why or why not? S mM vo (mmol ml-1
s-1) 1600 1.39 800 1.13 400 0.83 200 0.
54 100 0.32
Ans kcat 0.12 s-1 KM 470 mM
Yes.
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Previously defined Vo k2ES and ES
ES KM
Vo kcatES/KM
When SltltKM. E?ET
Vo kcatETS/KM
Kcat/KM rate constant for interaction of E and
S (turnover number) Can be used to measure an
enzymes preference for different substrates.
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New Kinetic Parameter
Kcat Vmax ET
when Kcat ltltS
Turnover Number
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Determining kcat and KM from intial rate data
o
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Vmax 150-160?? Km ???
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Lineweaver-Burk plot
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Vmax 150-160?? Km ???
Lineweaver- Burk plot
Vmax 164 mM/min Km 32.2 mM
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Figure 14-9 A double reciprocal (LineweaverBurk)
plot.
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Table 14-1 Values of KM, kcat, and kcat/KM for
Some Enzymes and Substrates.
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Figure 14-10 Cross section through the active
site of human superoxide dismutase (SOD).
Cu2 and R 143 form the binding site for O2-.
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Average 72.8 Standard Dev11.7
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Average 141 Standard Deviation 30
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A 170 B137-166 C 110-136