EXAM # 2 STUDY GUIDE

PROTEIN PURIFICATION
 Proteins will have different physical characteristics
depending on their AA sequence and length, this can
be used to purify a single protein to homogeneity
 Charged and/or polar AA are on the outside of the
protein and are charge dependent
 Crude Purification

SEPARATING PROTEIN MIXTURES

1. CHARGE
a. Proteins can have a variable range of pI
values; i.e. after two pH away from the PI,
then ???
b. Uses Ion-Exchange Chromatography:
i. Ion-Exchange Resins: DEAE
(diethylaminoethyl); weak anion
exchanger = creates an overall positive
charge and CM (carboxymethyl); weak
cation exchanger = creates an overall
negative charge
ii. high PI will elute with and high pH
and vice versa
2. SIZE
a. Uses size-exclusion chromatography
i. larger proteins will elute first, then
smaller molecules
3. AFFINITY
a. Uses FPLC (Fast Phase Liquid
Chromatography):
i. where the protein of interest attaches
to the stationary phase, and
contaminating proteins are washed off
using a mobile phase. Then a mobile
phase is used to wash off the protein of
interest.
b. Histidine Tag: the most common affinity
purification (6X-His Tag)
EXAM # 2 STUDY GUIDE

i. How to elute?
ii. Possible problems or complications?

SDS-PAGE AND ELECTROPHORESIS

 SDS-PAGE is used to visualize proteins
o This can be done using electrophoresis
 SDS (sodium dodecyl sulfate) is added
to denature the protein and coats the
surface and “brings out” a negative
charge which now scales to the size of
the protein—compared to known
protein weights.

PROTEIN-LIGAND INTERACTIONS
 Enzymes (catalysts) bind substrates, inhibitors, and
allosteric activators for regulation.
 Antibodies (immune proteins and made by B-cells)
bind virus, sugars, and other proteins.
 Motor proteins: actin, myosin, ATP
 Receptors recognize and sensor small molecules and
food sources, and react appropriately.
 Lectins: sugar-binding proteins
 Transcription factors: DNA (when to turn on and off a
gene) and RNA polymerase.
Protein-ligand interactions often cause conformational
changes via sensors.
 Bacteria would want to recognize maltose for sugar
catalysis for ATP, need for energy, and limited by
sugar supply.
 Binding is not lock-and-key, but more of an induced
fit; therefore, they are dynamic.
o Non-covalent H-bonds drive the
conformational change which helps respond to
the binding, thus, turning on certain signals.
EXAM # 2 STUDY GUIDE

o Enzymes also undergo conformational

changes upon substrate binding from subtle or
significant changes where chemistry occurs
 WHY MIGHT THIS BE
IMPORTANT?

MYOGLOBIN
A monomeric heme protein that binds and releases oxygen in
tissues; makes up about 2 mg/g of human muscle tissue, for
efficient delivery of oxygen to mitochondria.
 Deep-diving mammals have 10 to 30 fold more
myoglobin; this capacity for oxygen storage proteins
permits long periods underwater between breaths.
1. Structure and Function
a. Made up of one heme (an iron [Fe2+]
porphyrin) that binds oxygen.
i. apoprotein: without heme
ii. holoprotein: with heme
b. Oxidation catalysis or oxygen binding; heme
contains a central iron bound by 4 large
nitrogens (from a poryphyrin scaffold) leaving
2 open coordination sites for amino acids (His
or Cys) or ligands (O2)
c. Histidine is great at binding metal ions
(proximal; binding to the iron and distal;
stabilizes the bound oxygen via H-bonding)
d. The iron is protected by the plane of the
porphyrin ring system.
e. Binds O2 all the time by facilitating O2
diffusion in muscles because of very low
solubility O2 in blood; also acts as O2 storage.
2. Why is myoglobin good for oxygen storage?
a. Protein stabilization are at 2+ state via the
proximal histidine.
EXAM # 2 STUDY GUIDE

b. Iron spontaneously oxidizes to the 3+ state

which does not bind oxygen, thus, it can bind
and unbind oxygen quickly.
c. The iron can also bind carbon monoxide,
cyanide, and amines.
3. Why would myoglobin be bad for oxygen
transport from the lungs to tissues?
a. To efficiently transport oxygen from the lungs
to peripheral tissue, a transport protein needs
to be able to bind oxygen tightly (in the
lungs), but also be able to release it when
necessary (in tissue)
b. Myoglobin is a poor transport protein, but an
excellent storage protein due to its hyperbolic
binding curve.
4. Structure of myoglobin? (protein + heme)
a.
5. Structure of heme? Why use heme?
a. see below
b. Heme is used because Iron in the heme
spontaneously and rapidly oxidizes to the 3+
state in the open.
6. Coordination of iron cation? (roles of
proximal/distal His)
a. Fe2+ has six coordinating positions, four of
these are N’s of the heme, one is O 2, and the
sixth is the proximal histidine that stabilizes
the heme and the distal histidine that stabilizes
the bound oxygen via H-bonding
EXAM # 2 STUDY GUIDE

7. Why place heme inside of protein instead of using

free heme?
a. The structure of myoglobin suggests that the
oxygen-carrying heme group is buried inside
the protein portion of this molecule, which
keeps pairs of hemes group from coming too
close together. This is important, because
these proteins need to bind oxygen reversibly
and the Fe(II) heme, by itself, cannot do this.
When there is no globin to protect the heme, it
reacts with oxygen to form an oxidized Fe(III)
atom instead of an Fe(II)-O2 complex.
8. Carbon monoxide vs. Oxygen binding
a. Fe2+ porphyrins bind carbon monoxide 6000x,
however, myoglobin only binds CO 200x
stronger than oxygen.
i. The distance between the distal
histidine and the oxygen is shorter than
that of the carbon monoxide and distal
EXAM # 2 STUDY GUIDE

histidine, therefore, myoglobin has a

stronger affinity for oxygen.
9. Binding of Oxygen to Myoglobin
a. It is hard to measure O2 concentration in a
solution, but the oxygen partial pressure above
solutions can be measured, because pO2 is
roughly equal to [O2]; where P50 = oxygen
pressure at which half of the protein binding
sites are occupied, and YO2 = to the fraction of
sites occupied

[O 2 ] pO 2
YO2  
[O 2 ]  K d p O 2  P 50
i. P50 for myglobin is ~ 3 mmHg (tight
binding)
ii. PO2 in the arterial capillaries is 30
mmHg
1. Myoglobin won’t be good at
transporting at this point, but it
is a good bonding protein
iii. PO2 in the lungs is 100 mmHg

HEMOGLOBIN
1. Structure/Function – tetramer, alpha and beta
units
a. Tetramer: 2 alpha units and 2 beta units
i. Four heme group
b. O2 binding in lungs and releases in tissues,
where P50 is the PO2 where half of the binding
sites are occupied; T = low affinity state and R
= high affinity state
2. Contact points between sub-units
a. Hemoglobin has a quaternary structure with 4
peptide chains, with its own heme, are held
via non-covalent bonds; proteins, therefore,
EXAM # 2 STUDY GUIDE

are in contact with each other working in

concert.
b. Stationary and Mobile Areas
i. Strong interactions stabilize quaternary
structures in regions that do no move
—held by van der Waals and the AA’s
in the contact points are hydrophobic
ii. Mobile elements are held by salt
bridges, thus, ∆H is negative
(exothermic = making bonds) which
helps counterbalance the energy
needed to break the bonds
3. T-state vs. R-state (models)
a. Where would find these states? Which is
oxy/deoxy? Affinities for oxygen?
i. T-state would be found in low [O2],
thus it is deoxy; while R-state would
be found in high [O2], thus, it is oxy.
b. Transition from T to R state
i. Increasing the O2 concentration
induces a conformational change from
low affinity at low [O2] to high affinity
at higher [O2]
ii. Non-covalent inter-subunit interactions
are disrupted during the T  R
transition and the unfavorable enthalpy
loss is compensated by the favorable
O2-heme binding interactions
4. Changes in heme conformation – heme moving
back into plane, helix Fe2+
a. T  R States
i. T-State: Histidine helix shifts up
ii. R-State: O2 flattens out heme and
pulls Histidine and helix down with it
which results in helix shift
b. When O2 binds, it pulls the Fe2+ ion into the
plane of heme, causing steric strain between
EXAM # 2 STUDY GUIDE

the flattened heme and the proximal His (F8)

and Val FG5. Val FG5 is at the corner between
F and F helices. The strain is relieved by a
change in orientations of both His F8 and Val
FG5.

5. Cooperativity of oxygen binding – describe

concept of cooperativity and relate to model of
hemoglobin
a. A protein with a sigmoidal curve would
provide ideal binding characteristics and
hemoglobin has this via cooperativity form—
when the heme groups are communicating
together to gain or lose oxygen all together.
b. The ability of O2 to improve affinity for other
O2 molecules suggests cooperativity between
binding sites!
c. Cooperativity allows large scale changes in
binding with small only changes in pO2
EXAM # 2 STUDY GUIDE
Θ

6. Oxygen affinity of hemoglobin – compare with

myoglobin
a. hyperbolic vs. sigmoidal curve
7. Why is hemoglobin a good oxygen transporter?
a. hemoglobin is a good transporter because it
has a sigmoidal protein-ligand binding curve,
therefore, it has a high affinity for oxygen at
high concentrations and a low affinity for
oxygen at low concentrations; thus, it is able
to release oxygen easily relative to myoglobin
8. Allosteric regulation of hemoglobin?
a. Cooperativity implies that binding/release at
one site is going to result in a conformational
change at other binding sites that will facilitate
the binding/release of oxygen
i. ligand binding is regulating the
function of the protein at other binding
sites (allosteric regulation)
ii. homotropic: natural ligand causing
the change
iii. heterotropic: non-natural ligand
causing the change
b. Allosteric Transitions in Hemoglobin
EXAM # 2 STUDY GUIDE

i. KNF model/Sequential Model:

Binging oxygen at one subunit changes
conformation at that subunit,
facilitating transition at adjacent
subunits in the same molecules; mixed
tetramers with low- and high-oxygen
binding affinity subunits
ii. MWC model/Concerted Model: Hb
exists in two states; T(tense; lower O2
binding affinity) and R (relaxed; high
O2 binding affinity); O2 binding
perturbs the T  R equilibrium
toward the R state, and O2 release
favors the T-state; mixed tetramers are
not allowed in this model.
c. Quantitating Cooperativity = ???
9. Carbon Monoxide
a. CO sequester s heme which prevents oxygen
from binding
10. Bohr effect – pH and oxygen binding; source of pH
difference in lungs vs. tissues
a. Hemoglobin O2 binding is affected by pH,
where histidine (ionizable amino acid), an
acidic side chain, is involved. When O2 binds
with Hb, the proton is released and the pH is
decreased. As a result, P50 increases and the
curve moves to the right, thus, oxygen affinity
decreases and favors the T-state.
11. CO2 – carbamate formation
a. Oxygen consumed during cellular respiration
in aerobic tissue results in the release of
carbon dioxide. However, CO2 is not soluble
in water, instead it reacts with water to form
bicarbonate—a soluble buffer composed of
carbon dioxide and water.
EXAM # 2 STUDY GUIDE

b. When CO2 is transported as dissolved CO2 in

plasma to the lungs for exhalation, however,
5-13% is bound to Hb amino groups as
carbamate. The proton is releases, then it
contributes to the Bohr effect.

c. Consider Hyperventilation
i. Carbon dioxide levels decrease and
this favors the R-state and reduces
oxygen release in tissues; this is
treated by breathing into a paper bag to
reintroduce exhaled CO2 and increase
its concentration in the plasma, thus
favoring the low affinity T-state.
12. BPG and oxygen binding; high altitudes
a. Highly purified hemoglobin has a higher
affinity for oxygen that hemoglobin in whole
blood; thus, blood must contain some other
compound that affects oxygen binding to Hb
—metabolite BPG.
i. highly negatively charged and binds to
the + charged central cavity of the Hb;
this helps stabilize the T-state, thus,
releasing O2—absence of BPG has a
graph similar to myoglobin
13. Sickle Cell Disease
a. Describe mutation in hemoglobin causing
disease
i. The disease is caused by a single point
mutation (Glutamate/charged 
Valine/non-polar)
b. Polymerization of HbS
i. This mutation leads to a slight change
in structure of hemoglobin and
EXAM # 2 STUDY GUIDE

subsequent oligomerization and

insolubility, which decreases the
amount of active hemoglobin
c. Physiological effects of sickle cell
i. Because valine fits into EF pocket
(hydrophobic) of another hemoglobin
molecule, the abnormal erythrocytes
block circulation in the capillaries and
lyse due to their fragility, causing
anemia.
ii. Heterozygotes with half mutant and
half normal Hb are asymptotic except
when oxygen stressed
d. Why did sickle cell evolve?
i. HbS is prevalent in populations in
Africa since P. falciparum is unable to
infect sickled cells due to their shapes
and increased rates of phagocytosis of
infected sickle cells.
14. Quantitative descriptions of protein binding –
number of occupied binding sites
a. look at exercises in ppt or Mastering
Chemistry
15. Interpretation of ligand binding graphs
a. What do sigmoidal curves indicate?
i. A sigmoidal curve indicates a protein
that is able to hold oxygen at high
pressures and release oxygen at low
pressures, or when the body needs
more oxygen/less oxygen.
16. Magnitudes of Kd and relationship to binding
strength
a. Kd is the dissociation constant; the lower the
Kd, the higher the binding affinity; it is also
where half of the ligand is bound and the other
half is unbound
EXAM # 2 STUDY GUIDE

b. when Kon >>> Koff, the reaction is practically

irreversible
kof
MbO2 Mb O2
kon

k o ff [ M b ] [ O 2 ]
K d  
k on [M bO 2]

17. Do you understand the following relationships?

How do they relate to Hb affinity? Why is the
mechanism in which the alter Hb affinity?
a. Hb-BPG + O2  Hb-O2 + BPG
i. Happens in the lungs; the building of
oxygen favors the R-state, thus
releasing BPG, CO2, and H+
b. Hb-BPG + CO2  Hb-CO2 + BPG
c. Hb-BPG + H+  Hb-O2 + H+
i. The binding of BPG, CO2, and H+
favors the T-state, thus releasing O2
EXAM # 2 STUDY GUIDE

ENZYMES
 Enzymes are held by weak non-covalent bonds.
 Enzymes are catalysts
o increase reaction rates without being used up
o they are regenerated at the end of a reaction
 Most enzymes are globular proteins
o however, some RNA (rRNA and ribozymes)
also catalyze reactions
 ENZYMES ACCELERATE REACTIONS BY
STABILIZING THE TRANSITION STATE OF THE
REACTION!!!
o Therefore, enzymes do not change the Keq of a
reaction. Instead, enzymes change the rate of a
reaction
 Enzymes accelerate reactions by stabilizing the
transition state, i.e. lowering the ∆G; thus you can
write a well-recognized equation ∆G = ∆H – T∆S
o Enzymes can stabilize transition states by
improving either/or:
 the enthalpy of the reaction
 the entropy of the reaction
Properties of Enzymes
a. Reaction Rate Enhancements = Kcat / Knon
EXAM # 2 STUDY GUIDE

i. ratio that shows the magnitude of the

rate of the enzyme (how efficient an
enzyme is)
ii. 106 is fantastic for small molecule
catalysts
iii. catalysts do not change the
equilibrium; it speeds up the reaction
b. Reaction Conditions
Chemical Reactions Biological Reactions

Organic Water
Solvents (harsh)
Heat it up to 100 deg C 37 deg C

Oxidized O2

Pressure 1 ATM

c. Regiospecificity
i. isomer specificity; one structural
isomer is produced exclusively when
other isomers are also theoretically
possible
d. Stereospecificity
i. stereoisomeric specificity; a reaction in
which the stereochemistry of the
reactants controls the outcome of the
reaction; i.e. one stereoisomer of a
certain reactant produces one
stereoisomer of a certain product,
whereas a different stereoisomer of the
same reactant produces a different
stereoisomer of the same product.
e. Geometric (Substrate) Specificity
i. Regiospecificty + Stereospecifity
ii. Enzymes bind to their substrates
selectively and in a defined orientation
1. Describe the models for ligand binding:
EXAM # 2 STUDY GUIDE

a. Lock and Key: The specific action of an

enzyme with a single substrate
b. Induced Fit: assumes that the active site of an
enzyme is not complementary to that of the
transition state in the absence of the substrate.
Such enzymes will have a lower value of
Kcat/Km, because some of the binding energy
must be used to support the conformational
change in the enzyme. Induced fit increases Km
without increasing kcat.
2. Most enzymes are globular proteins – require
native fold for catalytic activity
a. Globular proteins are only marginally stable
because the free energy released when the
protein folded into its native conformation is
relatively small. This is because protein
folding requires entropic cost. As a primary
sequence of a polypeptide chain can form
numerous conformations, native globular
structure restricts its conformation to a select
few. It results is a decrease in randomness,
although non-covalent interactions such as
hydrophobic interactions stabilize the
structure.
3. Why use enzymes as catalysts?
a. because enzymes increase the rate of virtually
all the chemical reactions within cells; it
would take millions of years for certain
reactions to take place if enzymes weren’t
used.
4. Draw and label generic reaction coordinate
diagram and reaction coordinate diagram for
enzyme/substrate complex for
catalyzed/uncatalyzed reactions.
EXAM # 2 STUDY GUIDE

5. Using proximity
a. Entropy: Proximity
i. If two reactants need to reach with
each other in the solution, they have to
be able to find each other. As a
solution, enzymes have sites that
selectively recognize reactants and
bring them together.
ii. Proximity and orientation favor
formation of the transition state
∆Scat > ∆Snon
b. Entropy: Orientation
i. Reagents need to react at just the right
geometry for a reaction to occur; thus,
enzymes orient the reagents so they are
in conformation that favors the
reaction
6. Transition state stabilization
a. Enzymes enforce a change in the shape of the
substrate to look more like the transition state;
essential van der Waals interactions
EXAM # 2 STUDY GUIDE

i. weak interactions between the enzyme

and the substrate are optimized in the
transition state
ii. binding of substrates and intermediates
in the enzyme active site are
complementarity (chemical and shape)
between enzyme and
substrate/intermediates/transition
state(s) has to be “fine-tuned”—simply
binding substrates tightly doesn’t
translate to better catalysis
iii. To lower the activation energy, the
system must acquire an amount of
energy equivalent to the amount by
which ∆G is lowered (∆GG); much of
this energy comes from the binding
energy contributed by the formation of
noncovalent interactions between
“substrate” and enzyme in the
transition state that are stronger than
the interaction of enzyme with ground
state of the substrate.
b. Stickase Example
i. Uncatalyzed (Knon)

ii. Enzyme Complementary to the

Substrate: the enzyme is not
complementary to the transition, thus,
the free energy of ES is much lower
and the free energy of the TS doesn’t
change. RESULT: activation energy
(∆G) is much higher in the absence of
EXAM # 2 STUDY GUIDE

“catalyst”, so the rate constant would

be dramatically decreased.

iii. Enzyme Complimentary to the

Transition State: Active site is much
more complementary to the transition
state than to substrates, so the enzyme
binds to the transition state much more
tightly than it binds substrate—free
energy of TS is reduced, but the free
energy of ES isn’t much different from
the free energy of E + S. RESULT:
activation energy is dramatically
decreases, thus, the rate constant is
increased!

7. Acid/Base Catalysis
a. GABC (General Acid/Base Catalysis)
i. Titratable amino acids provide or take
protons away depending on the needs
of the reaction
ii. His, Asp, Glu, Lys, Cys, Tyr, Arg; H +,
partially charged amides, and metals
(2+) can also serve this function
b. Enzymes avoid unstable charged
intermediates in a reaction (which would have
high free energies) by having groups
EXAM # 2 STUDY GUIDE

appropriately located to donate a proton or

accept a proton.
i. If a group donates a proton (acts as a
general acid) in a chemical
mechanism, it has to get a proton (a
different one) back (acts as a general
base) by the end of the catalytic cycle,
and vice cersa.
ii. Obviously, pH influences state of
protonation of enzyme functional
groups, so catalytic activity of
enzymes using GABC is sensitive to
pH.
8. Covalent Catalysis/Nucleophilic Analysis
a. Changing reaction pathway through formation
of an enzyme-substrate covalent intermediate
(a transient formation of a catalyst-substrate
covalent bond)
i. His, Cys, Asp, Lys, and Ser can
participate in covalent catalysis by
acting as nucleophiles
ii. see Chymotrypsin
9. Metal-ion Catalysis
a. metal ions are often used for one or more of
the following
i. binding substrates in the proper
orientation
ii. mediating redox reactions
iii. electrostatically stabilizing or shielding
negative charges
b. metalloenzymes contain tightly bound metal
ions
c. metal-activated enzymes contain loosely
bound ions
d. some prosthetic groups are metalloorganic
compounds
10. Electrostatic Catalysis
EXAM # 2 STUDY GUIDE

a. when substrate binds to enzyme, water is

usually excluded from the active site
(desolvation)
i. causes a local dielectric constant to be
lower, which enhances electrostatic
interactions in the active site
ii. leads to protection of the reactive
groups from water, so water doesn’t
react to form unwanted bi products;
water does come in if it’s a substrate,
but only in a certain sub-part of the
active site
b. Involvement of charged enzyme functional
groups in stabilizing otherwise unstable
intermediates
c. Charge build-up during a reaction should be
neutralized by the opposite charge
i. Arg, Lys, and His neutralize a negative
charge
ii. Asp and Glu neutralize a positive
charge
11. Catalysis increase reaction rates by lowering
activation energy

MICHAELIS-MENTEN KINETICS (see another sheet)

1. Initial Velocity and Max Velocity
2. Initial velocity and max velocity
3. Steady state assumptions
4. Derivation of M-M equation
5. Michaelis constant (Km) and use
6. Representing MM kinetics
7. Relationship of Km to Kd, Kcat, turnover

CHYMOTRYPSIN STRUCTURE AND FUNCTION

 an example of a serine hydrolases (including serine
proteases) are a broad class of enzymes that use a
EXAM # 2 STUDY GUIDE

serine nucleophile to cleave ester or amide bonds

(also called “endoproteases”)
1. Is this enzyme specific for certain substrates?
a. It cleaves on the C-terminal of phenylalanine
(F), tryptophan (W), and Tyrosine (Y) on
peptide chains. It shows specificity for
aromatic AA because of its hydrophobic
pocket.
2. What reaction does it catalyze?
a. Chymotrypsin is expressed as a single
polypeptide that self-cleaves to produce the
active enzyme that enhances the rate of
peptide bond hydrolysis by a factor of at least
109
3. Catalytic triad – what residues are important in
the reaction? What is their role?
a. serine hydrolases (like the protease
chymotrypsin) have an active site catalytic
triad: Asp – His – Ser which are linked by low
barrier hydrogen bonds that facilitate the
deprotonation of the serine; as a result, serine
can now act as a nucleophile to facilitate
cleave of the amide (proteases) or ester
(estrases) bonds
i. effective pKa of the serine side chain
is ~7
4. What category of catalysis does this reaction fall
under?
a. Covalent Catalysis
5. Two distinct phases – acylation and deacylation
a. Acylation: the first half of the reaction—the
enzyme itself provides a potent nucleophile, a
specific Ser OH group (which is made more
nucleophilic with the help of a nearby His
residue that acts as a general base/proton
acceptor)
EXAM # 2 STUDY GUIDE

i. Catalytic serine attacks the peptide

bond—carbonyl pi-electrons move onto
oxygen; this attack produces a covalent
bound tetrahedral intermediate (Covalent
Catalysis)
ii. Collapse of oxygen electrons back
onto the carbonyl releases the peptide
amine fragment as a leaving group; the
departing amine must grab a proton,
which it does from the Histidine that
deprotonated the serine (GABC)
iii. oxy-anion hole: the buildup of
negative charge on the oxygen is
destabilizing in the transition state which
is stabilized by the oxy-anion whole.
1. Oxy-anion hole preferentially
binds O- of a tetrahedral (sp3)
intermediate using H-bonds
from the partially positive N-H
groups, however, the carbonyl
oxygen is planar (sp2);
therefore, the oxyanion hole is
organized in such a way to
stabilize both the charge and
shape of the TS/intermediate
(not the starting material)
EXAM # 2 STUDY GUIDE

2. Serine-195 can attack boronic

acids (sp2)/contain an empty
orbital and the oxygen in serine
can add to the empty orbital
which then generates a
covalently bound, negatively
charged, tetrahedral complex
(B- = acts as an analog)

b. Deacylation: a second substrate, H2O, enters

and acts as a nucleophile, attacking the
carbonyl C of the acyl enzyme (carboxylate
ester), hydrolyzing the bond to displace the
second product, the N-terminal portion of the
original substrate, with its new C-terminal a-
carboxyl group, and regenerating the alcohol
component (the enzyme with its Ser-OG free
again); the carboxylic acid “half” of the
original peptide/protein is released.
EXAM # 2 STUDY GUIDE

i. Once the cleaved amine-containing

peptide departs, water can come orient
itself in the space once occupied by the
peptide; which is then deprotonated
(GABC) by the His, creating an –OH
nucleophile that can attack the stable
acyl-enzyme complex.
ii. Water attacks and regenerates a
negatively charged tetrahedral
intermediate (stabilized by the
oxyanion hole), and the collapse of a
negatively charged oxygen release the
peptide, and the serine grabs its proton
back from His to regenerate the
starting enzyme.
6. Learn the mechanism. Be able to explain the role
of the residues of the catalytic triad; the
importance of the enzyme tertiary structure
(hydrophobic pocket/oxyanion hole adjacent to
active site).

LYSOZYME STRUCTURE AND FUNCTION

1. Is this enzyme specific for certain substrates?
What are they?
EXAM # 2 STUDY GUIDE

a. cleaves glycosidic linkages of a

polysaccharide located on the cell wall of
bacteria
i. lyses the cells – hence lysozyme; a
deep cleft allows the enzyme to access
the polysaccharide
2. What reaction does this catalyze?
a. it catalyzes the hydrolysis of the 1  4 linkage
between NAM and NAG
3. What would happen if a patient lacked lysozyme?
a. It would interfere with the cells ability to
remove/clear damaged or unnecessary cellular
components which could be
toxic/carcinogenic/mutagenic due to by-
product build-up.
4. Describe catalytic mechanism of lysozyme.

a. The Role of the Strain: Early modeling

suggested that the D-ring would have to take a
half chair like configuration to fit in the active
site: The role of the strain (transition state
shape) in lysozyme catalysis.
EXAM # 2 STUDY GUIDE

i. Cleavage of the glycosidic bond is

facilitate by electron donation of the
endocyclic oxygen atom which helps
push off the leaving group; this leads
to the formation of a double bonded
oxocarbenium ion intermediate (half-
chair configuration).
ii. The enzyme preferentially binds the D-
ring in a half chair conformation; the
oxocarbenium transition states must be
in a half chair conformation due to
double bond planarity, therefore, the
enzyme preferentially binds the shape
of the transition state—not the
substrate.
b. GABC and Electrostatic Catalysis:
i. Two titratable amino acids, E35 (Glu
E35) and D52 (Aspartate f2), are
located next to the cleaved bond
1. Glu35 acts as a proton donor to
the oxygen of the leaving
alcohol
2. The resulting carbonium ion
(+) is stabilized by the ionized
side chain of Asp 52 (via
electrostatic catalysis) until it
can react with water.
c. Covalent Catalysis
EXAM # 2 STUDY GUIDE

i. was introduced by Withers, et. al. in

2001 as an alternative mechanism
(green arrows)