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PROTEINS AND ENZYMES
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The Amino Acids
Basic Protein Structure
Hemoglobin, Myoglobin, Oxygen Transport
Collagen
Enzymes
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THE AMINO ACIDS
NAME
SYM
GLYCINE
Gly
STRUCTURE
R-GROUP
GROUP /
OTHER
Proton
NONPOLAR,
ALIPHATIC
G
The only
NON-CHIRAL
amino acid.
Minimal
hindrance
allows much
more
structural
flexibility.
ALANINE
Ala
Methyl
NONPOLAR,
ALIPHATIC
Isopropyl
NONPOLAR,
ALIPHATIC
Isobutyl
NONPOLAR,
ALIPHATIC
A
VALINE
Val
V
LEUCINE
Leu
L
ISOLEUCINE
Ile
sec-Butyl
I
PROLINE
Pro
P
NONPOLAR,
ALIPHATIC
Not that it has
TWO CHIRAL
CENTERS -- at
alpha-Carbon
and at first
Carbon of
chain.
Cyclopentyl
Amine
NONPOLAR,
ALIPHATIC
Rigid
configuration
= less
structural
flexibility.
Only a.a. with
a 2 amino
group. Side
chain is
covalently
bonded to the
nitrogen of
the amide.
Often found at
beta-turns
(corners) of
beta strands
and sheets
SERINE
Ser
1 Alcohol
POLAR,
UNCHARGED
2 Alcohol
POLAR,
UNCHARGED
S
THREONINE
Thr
T
CYSTEINE
Cys
C
Also has TWO
CHIRAL
CENTERS.
Thiol
POLAR,
UNCHARGED
Readily
oxidized to
form cystine
= two
cysteine
molecules
joined by a
disulfide
bridge.
METHIONINE
Met
M
ASPARAGINE
Asn
N
Sulfur Ether POLAR,
UNCHARGED
Cannot form a
disulfide
bridge.
Amide,
POLAR,
connected at UNCHARGED
alphaThe amide of
Carbon
aspartate.
The shortest
of the two
a.a.'s
containing
amides in the
side chain.
GLUTAMINE
Gln
Q
PHENYLALANINE Phe
F
TYROSINE
Tyr
Y
Amide,
POLAR,
connected at UNCHARGED
beta-Carbon
The amide of
glutamate.
Toluene
AROMATIC
A phenyl
group is
substituted
for one of the
H's of alanine
-- hence the
name.
para-Methyl- AROMATIC
phenol
Crystalline
substances
found on
crusty cheese.
Named for
"Tyros," Greek
God of
cheese.
Can form
hydrogen
bonds.
Important in
enzymatic
activity -tyrosine
cascade.
More polar
than
phenylalanine.
TRYPTOPHAN
Trp
AROMATIC
W
More polar
than
phenylalanine.
Strongly
absorbs
ultraviolet
light.
Only bicyclic
side-chain.
Known as an
INDOLE
structure.
LYSINE
Lys
pKa 10.
K
Butyl amine POSITIVELY
CHARGED
(BASIC)
If pH of
environment
< 10, the
group is
positively
charged.
If pH > 10,
the group is
neutral.
ARGININE
Arg
pKa 12: MOST
BASIC of all
amino acids.
R
Guanidino
Group
POSITIVELY
CHARGED
(BASIC)
Arginine is
almost always
positively
charged. No
biological
environment
is basic
enough to
neutralize it.
HISTIDINE
His
pKa 7
H
Imidazole
group
POSITIVELY
CHARGED
(BASIC)
It's charge is
specifically
controlled by
the pH of its
biological
environment.
ASPARTATE
Asp
ASPARTIC ACID D
pKa 5 for the
SIDE CHAIN.
NEGATIVELY
CHARGED
(ACIDIC)
At pH < 5, it is
acidic (with
COOH) and
called
aspartic acid.
pKa = 2-3 for
the PEPTIDE
CHAIN, as it
usually does.
At pH > 5, it is
in form
COO-and
called
aspartate.
GLUTAMATE
Glu
GLUTAMIC ACID E
pKa 5 for the
COOH SIDE
CHAIN.
pKa = 2-3 for
the PEPTIDE
CHAIN, as it
usually does.
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NEGATIVELY
CHARGED
(ACIDIC)
At pH < 5, it is
acidic (with
COOH) and
called
glutamic
At pH > 5, it is
in form COOand called
glutamate
BASIC PROTEIN STRUCTURE
Classifications of Proteins:
Globular
Fibrous
Lipoproteins
Nucleoproteins
Glycoproteins / Proteoglycans
Ways to show a protein:
Chemical (molecular) structure.
Ball and stick model.
Space-Filling Model: Based on the Van-der-Waals maximum radii each
nucleus would occupy.
Chirality: alpha-Carbon is chiral, in all amino acids except Glycine.
L-forms of amino acids are the kinds that are found in nature.
Other Amino Acids not found in proteins:
GABA: Gamma-Amino Butyric Acid.
Ornithine -- metabolic intermediate
Homocysteine -- vitamin metabolism intermediate
Homoserine
Thyroxine -- catabolic hormone from thyroid gland, derived from tyrosine.
Weak Acid / Acid Equilibrium Constant:
Henderson Hesselbach Equation:
Tyrosine as a weak acid:
pKa1 = 2.2
Carboxylate dissociation: COOH ------> COO- + H+
So, at pH of 2.2, 50% of the COOH moieties are dissociated.
pKa2 = 9.1
Amino dissociation: NH3+ ------> NH2
pKa3 = 10.1
Phenol hydroxyl group ------> phenoxide
So, at biological pH, it has a net charge of zero (COO-, NH3+, and OH)
Isoelectric Point: The point at which the net charge on the protein is zero, and
the concentration of zwitterion is at its highest.
Ultraviolet Absorption of Proteins:
Aromatic amino acids show up on UV-Absorption spectra: Tryptophan is the
strongest signal, and Phenylalanine the weakest. Tyrosine in the middle.
Post-Translational Modifications:
Cystine: The oxidized form (disulfide bridge) of two cysteines.
Many other examples... collagen, glycosylation, etc.
Structural Analysis of Proteins:
Primary sequence can be determined by the Edman Degradation.
Individual amino acids can be separated by ion-exchange, high-pressure
liquid (HPLC), and gas chromatography.
Can predict from nucleic acid sequence.
Clinical Example of Too Little Protein: Spherocytosis
Decreased level of the erythrocyte anion transporter, Band-3, leads to
spherocytosis. Biconcave shape can no longer be accomplish by
erythrocytes.
Lack of the anion-transporter leads to reduced osmotic capabilities
Polyacrylamide Gel Electrophoresis: PAGE gel, separating proteins after
detergent with Sodium Dodecyl Sulfate (which denatures the protein).
Then move them on an electrical sieve: polyacrylamide gel.
The smallest proteins will travel the fastest (i.e. the furthest).
Clinical Example of Too Much Protein: Myeloma
Light Chain Deposition Disease: Too much of the mappa (small) chain of
immunoglobulins. Proliferation of the plasma cells that make this single
chain globulin.
Immunoglobulins consists of four subunits, but they are not quaternary,
because they subunits are covalently linked to each other.
Primary Structure: The sequence of amino acids.
Secondary Structure: Alpha-Helices and Beta-sheets. The interactions and
special orientations of neighboring amino acids.
Supersecondary Structures: Locally folded domains. The folding in local regions
of a protein.
Locally folded domains are repeated in discrete proteins throughout the
genome. Similar motifs (and repeating motifs) arise in many different
proteins, indicating repetition of gene sequences in evolution.
Tertiary Structure: The folding and three-dimensional shape of entire proteins,
resulting from covalent and non-covalent interactions between regions.
Quaternary Structure: The non-covalent (physical) interactions between
different monomers of a polymeric protein, such as hemoglobin (4 subunits) and
tubulin (2 subunits).
Techniques for Structural Analysis of Tertiary Conformation:
X-Ray Crystallography: Must form stable crystals or possess repeating
structural patterns in order to visible by this method.
Magnetic Spectroscopy: NMR-Spectra. C-13 and H-1 absorption of radio
frequencies in a magnetic field.
Heme Group: Coordinate ligands form, usually with Fe in the middle. The best
example of a prosthetic (non-amino-acid) group in a globular protein.
Cytochrome-C contains a Heme-group. It interacts with a membrane. Since
the membrane is negatively charged, it has positively charged Lysine
groups to achieve the interaction.
The positively charged Lys residues are highly conserved across
species, in the same 3 position.
It is a porphyrin ring, as well as an Iron.
Iron has six coordination points
Four of them contributed by the porphyrin ring.
Two of them are sulfur atoms contribute by Methionine and Nitrogen
contributed by Histidine.
Non-Covalent Interactions: Important to secondary and tertiary structure
Electrostatic Forces: +/- attraction
Hydrogen Bonding: OH, NH, SH
Hydrophobic Interactions: Physical attraction between hydrophobic
moieties in water.
Van der Waals Forces: Weakest, interactions relative to size.
Peptide Bond: A Condensation Reaction, formed by the exclusion of water
between two amino acids.
The peptide bond is rigid and planar.
It is rigid because it has double-bond character (resonance) resulting from
the partial negative charge of the nitrogen and carbonyl (alpha,betaunsaturated characteristics).
Phi (phi): The angle between each nitrogen and the alpha-Carbon.
Psi (): The angle between each alpha-Carbon and the Carbonyl-carbon
Alpha Helix:
3.6 residues make up each complete helical turn.
It is a right-handed helix.
Electrostatic interactions (charged residues) are uncommon, and when they
are present they mutually cancel or attract each other to stabilize the turn.
Beta-Strand: Form an approximate plane.
Beta-Sheet: Adjacent beta strands, in parallel or anti-parallel orientations.
Turns: The point where the strands of a beta-sheet may change direction.
Glycine is often found in turns, due to small size and no steric
hindrance.
Proline if often found in turns, due to its secondary alpha-carbon and
natural angle for bending.
The R-Groups in beta-strands point away from the plane, usually in
alternating up and down directions.
Ramachandran Plot: Plot of Psi -vs- Phi angles, showing what theoretical values
of Psi and Phi are possible between residues, and what secondary conformations
they dictate.
Hypercholesterolemia: Results from the single-point mutation of Apo-B (gigantic
protein), from Gly ------> Val. Gives us 2 to 4 times too much LDL (low density
lipoprotein) cholesterol is a result.
They think it screws it because Gly is at a key turn, and Val is too bulky to
allow the turn to remain... changes tertiary conformation.
Primary Structural Analysis: Strategy of putting puzzle pieces together
Digest (oxidize) cystine bonds to cysteic acid.
Digest with Trypsin, which cuts at specific residues.
Cleaves at the Carboxyl terminus of Lys and Arg.
Digest with Cyanogen Bromide, which cuts at different specific residues.
This cleaves at the Carboxyl terminus of Methionine.
So, if you cut one chain into two pieces, then you know that Met was
in the middle of the chain. If, on the other hand, you get one piece,
then you know that Met was at the end (C-Terminus) of the chain.
Align peptides with overlapping sequences from the two above.
Protein Chaperones (Heat Shock): Proteins that aid proteins in folding and
unfolding. They unfold proteins so that they can get through a membrane intact,
and then refold them on the other side.
Fluorescent Spectroscopy: Identification technique to tell the relative abundance
of the aromatic amino acids. It will distinguish between a polar and non-polar
environment for these residues.
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HEMOGLOBIN, MYOGLOBIN, OXYGEN TRANSPORT
Hemoglobin: Oxygen affinity
Oxygenated blood has Oxyhemoglobin, which holds four oxygens each
bound to an iron in the Heme group.
Deoxygenated blood has Carbaminohemoglobin (carbamate). Two CO2's
can be held by each hemoglobin. The rest is dissolved in plasma as HCO 3-.
Myoglobin: Similar to hemoglobin, it has even higher affinity for O2 (and can take
O2 from Hb). Found in skeletal and cardiac "red" muscle.
Made up of 75% alpha-helices. The rest is random coil. Predominately
hydrophobic. Non-polar in order to...
In order to bind solubilize non-polar O2
In order to bind the non-polar Heme group.
Protein can be divided into 8-segments (A-H)
Heme group has one (not four) Iron atoms.
It is a ring system, mae mostly of carbon atom. The four atoms
closest to the iron are nitrogens.
Porphyrin Ring forms an almost exact plane.
In deoxy-Mb, the Fe sits above the plane. It is pulled out of the plane
by the proximal Histidine (His-93), the 5th coordinating ligand.
The rest of the protein minimizes the oxidation of Fe -- tries to keep it
in Fe+2 form, which bins O2 much better than Fe+3.
Oxygenation of the Myoglobin does not change its structures very much, as
compared to hemoglobin. Only 3 residues swing out of the plane a little bit.
The Mb molecule has dynamic configurations: (1) it opens up a little
to let the O2 in, then (2) it clamps down to "hold on" to the O2.
The Bohr Effect, CO2 / Hemoglobin Interactions:
Carbonic Anhydrase: The enzyme that catalyzes CO2 + H2O ------> HCO3- + H+,
to store CO2 in plasma.
Both HCO3- and H+ are picked up by the deoxyhemoglobin.
The sloughed off HCO3- and H+ go back to CO2 + H2O in the lungs
(compliments of carbonic hydrase), and the CO2 is expelled.
Oxygen Dissociation Curve: A graph of the Partial Pressure of O2 in the blood
-vs- the Hb-Saturation -- the percentage of Hemoglobins that have O 2 bound.
P50 = the point of half-saturation. For myoglobin that is around 2 or 3
partial pressures O2, which is pretty low, which means that myoglobin can
bind O2 at low partial pressures.
Because of myoglobin's high O2-affinity, it would not be a good
O2-transporter, because it wouldn't release O2 into the tissues.
Sigmoid (S-Shaped) Curve: For O2-Dissociation, it shows weak binding in
tissues and strong binding in lungs.
Proteins that show S-Shaped curves have multiple binding sites (as in
Hb).
Hill Coefficient: There is cooperation between the multiple binding sites.
The degree of cooperation is quantified by this coefficient. It is a measure
of that degree to which one bound site promotes the binding of further
sites.
For Hb the hill coefficient can have a value from 1 to 4. For Hb, it is
usually 3.
For Myoglobin, the Hill Coefficient value is 3, indicating no
cooperation.
Oxygen Saturation: Different ways of calculating Y
Y = (Occupied ligand binding sites) / (total binding sites)
Y = (Oxygenated Myoglobins) / (Total Myoglobins)
Hemoglobin Structure:
Hb consists of 4 subunits
alpha1beta1 exist as a "dimer" subunit. alpha2beta2 exist similarly.
They are each called heterodimers.
Variability can be accommodated in the beta-structures as long as
certain critical residues remain the same.
T-State: Deoxy Hemoglobin exists in the T-State. "Tense, tight, taut."
Non-covalent interactions between the two heterodimers is strong.
The cavity between the beta-subunits is large.
R-State: Oxy-Hemoglobin. Relaxed.
Bohr Effect: Negative effectors that decrease O2-affinity
Lower pH: Shifts the O2-dissociation curve to the right.
More CO2: Shifts the O2-dissociation curve to the right.
2,3-bisphosphoglycerate: Shifts the O2-dissociation to the right.
It is a polyanion. The deoxy form (larger beta-cavity), which is a
positively charged center more readily accommodates the multiple
negative charges.
Bohr Effect: Positive effectors that increase O2-affinity.
Oxygen: Shifts the O2-dissociation curve to the left.
The presence of O2 facilitates the binding of more O2!
Hemoglobin-F: Fetal Hemoglobin
Has a higher affinity for O2.
Structure = alpha2gamma2. The gamma-chain differs from the beta-chain
by only one amino acid.
Replace His143 with Ser, resulting in less negative charge ------> less
affinity for BPG ------> Curve shifts to the left.
Mutant Hemoglobins: If the mutation is harmful, then it is an hemoglobinopathy.
However, many Hb mutations are "silent."
Methemoglobin: Oxidized Ferrous ------> Ferric. Result = hypoxia.
Symptom = Cyanosis.
Shepherd's Bush: Hear the curve shifts to the left ------> Too high of an
affinity for O2
Polycythemia = too many red blood cells in circulation. This is the
result. More red-blood cells (hence a larger denominator in the
O2-saturation value) means a lower overall saturation. So this is a
compensatory symptom.
Hammersmith: Heme group can no longer bind the beta-chains due to a
point-mutation of Phe ------> Ser, which leads to less hydrophobicity.
Only two O2-binding sites as result.
Bibba: Dissociation of the tetramer.
Hemoglobin Kansas: Found a mutation on half of the beta-chains in this
case. It affected the interaction in the contact regions between alpha 1 and
beta2.
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COLLAGEN
Hallmark Structure of all Collagens: Triple Helix. There are different types, use
SCAB to remember:
S = SKIN = Type I Collagen
C = CARTILAGE = Type II Collagen
A = Aorta (vessels) = Type III Collagen
B = Basal Membrane = Type IV Collagen
Collagen Types can be identified by the chains that constitute them, ex.
Collagen I consists of (Alpha-1-I)2(Alpha-2-I)
Diseases Caused by Collagen Mutations:
Ehlers Danlos Syndrome (EDS): A group of disorders characterized by
laxity of joints, skin abnormalities, arterial aneurysms. Mutations in the
synthesis of collagen (not necessarily structure)
EDS VII: Mutation alters the cleavage site in Type Procollagen (with
end-tails still attached). Prevents by cleavage by Procollagen
N-Proteinase. Accumulation of procollagen alters fibril formation.
EDS IV: Mutation in Type III Collagen.
EDS VI: A deficiency where cross-links cannot be made due to
mutated lysyl hydroxylysine enzyme.
Osteogenesis Imperfecta: Mutation of Type I Collagen, causing brittle
bones.
Structurally defective pro-alpha-chains of Type I, which interfere with
folding of the triple helix (3 conformation) or with fibril formation.
Alport Syndrome: Type IV mutation. Problems in the ears and kidneys.
Autoantibodies targeted to type IV.
Goodpasture Syndrome: Acquired disorder in which patient develops
antibodies against their own collagen.
Structure of the Collagen Chain:
One collagen chain is a triple helix, composed of three sub-chains,
consisting of a total of about 1000 amino acids.
Hydroxyproline: Collagen chains all have the OH group added on Proline
residues.
Hydroxylysine: College chains all have OH group added to Lys residues.
Contain Fructose disaccharide (Glucose + Galactose), hooked onto the
Lysine residue. Glycosylated Lysines are unique to all collagens.
Every third residue is Glycine. It is the smallest, and it is the only residue
that can fit in the pocket of the helix. Any point mutation replacing Gly will
cause a kink in the chain and change the properties of the collagen.
Tropocollagen: A soluble form of collagen that must be further processed to form
a fiber.
It will form fibrils by using the quarter-stage overlap (three fourths of each
helix chain overlapping).
collagen has a lot of post-translational modifications. One of them is the
formation of cross-links between chains. The cross-links are formed
between lysine and hydroxylysine. Hydroxylysine is essential to crosslinking.
Hyperextensibility of the skin: Failure to form cross-links due to no
formation of hydroxylysine.
Biosynthesis of Collagen:
Procollagen: The form of the individual chains inside the cell. They are
soluble and still have their tails.
Procollagen has end-pieces called propeptides.
Tropocollagen: Outside the cell, the procollagen is first converted to
tropocollagen. Once the propeptides are gone, self-assembly of the
procollagens occurs.
The tropocollagen is then converted to a fiber, and then to a cross-linked
fiber.
Prolyl Hydroxylase: Enzyme that converts proline to hydroxyproline.
This enzyme requires Vitamin C! Scurvy, Vit-C deficiency, results from
no hydroxyproline.
Fe+2, O2, and alpha-ketoglutarate are also needed. Vitamin-C serves
to keep Fe+2 in the reduced (+2) state.
Lysyl Hydroxylase: Enzyme converts lysine to hydroxylysine.
Requires the same cofactors as prolyl hydroxylase.
Lysine and Hydroxylysine, again, are the precursors of the cross-links.
Lysyl Oxidase: The enzyme that forms that cross-links between Lysine and
Hydroxylysine.
It converts the OH groups on the hydrolysines to aldehydes. They
then form cross-links, aldol condensations, with other aldehydes.
Osteogenesis Imperfecta: Revisited. Type I Collagen defects.
One of the disorders is related to a shortened chain (deletion). Then the
mutated chain is shorter than the other two, ruining the whole structure.
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ENZYMES
Osteoarthritis: Collagenase is not repressed during adult life, resulting in
digestion of collagen in joints. Collagenase is only supposed to be active during
development for reshaping of collagen.
Rheumatoid Arthritis: Tissue proliferates and invades the joints.
Cancer Metastasis: Proteolytic Enzymes are required for cancer cells to
metastasize, as they must degrade the basement membrane of the origin tissue
to get through to the lymph system, and degrade the basal membrane of the
target tissue to get into it.
Phenylketonuria: A disorder caused by mutation in the enzyme phenylalanine
hydroxylase, which converts phenylalanine to tyrosine.
Thermodynamics:
First Law: Energy is neither created nor destroyed. It only changes form.
DeltaE = Q - W = (Heat absorbed by system) - (Work done by system)
This only applies to the system. Energy can be lost to / taken
from surroundings.
DeltaETOT = DeltaESYS - DeltaESURR
Second Law: A process occurs spontaneously only if the sum of the entropy
in the system and its surroundings is greater than zero. Entropy always
increases.
1M NaCl diffusing through membrane to create a partition with 0.5M
on either side is an example of increasing entropy, increasing
randomness.
Gibbs Free Energy: DeltaG = DeltaH - TDeltaS
DeltaG provides no info about the rate of a reaction. That depends on
the free energy of activation, DeltaG.
Zero-Order Kinetics: Rate of the reaction is constant.
Rate = k
First-Order Kinetics: Rate of the reaction is dependent on concentration of one of
the substrates
Rate = (k)[Reactant]
Concentration of Reactant, [A]: At any time, it is equal to (rate of reverse
reaction) - (rate of forward reaction)
Second-Order Kinetics:
Rate = k[A][B]
When B is much larger than A, B is no longer limiting the reaction, and it
appears to be dependent on [A]. This kapparent.
Standard Free Energy: How to get standard free energy from the K eq
Free Energy of any particular reaction:
Relation between Free Energy of Activation and K: Increasing the activation
energy causes an exponential decrease in the rate constant.
Ground State: The lowest energy and most stable form.
Transition State: The highest energy and least stable form.
Classifications of Enzymes:
Oxireductases: Enzymes that swap electrons / do redox reactions.
Transferases: Enzymes that transfer functional groups to another
component.
Hydrolases: Cleavage (hydrolysis) using water.
Lyases: Cleavage of double-bonds (reduction).
Isomerases: Converting the reactant from one isomer to a different isomer.
Ligases: Join structures together.
Cofactor: A non-enzyme component of a reaction that is required for the reaction
to run. Usually a vitamin or mineral. Cofactors are usually easily separated from
their enzyme.
Coenzyme: A cofactor that is also itself an enzyme.
Prosthetic Group: Cofactors that are more tightly bound to an enzyme, usually
covalently, as in the heme group of hemoglobin.
Unique Features of Enzymes:
Efficiency: Either much faster, or in the case of collagenase, slower than the
reaction would otherwise be.
Binding Specificity
Allows reactions to occur under physiological (mild) conditions.
Catalytic activity may be regulated by other enzymes or the physiological
environment.
ACTIVE SITE: The part of the enzyme where the reaction takes place.
It is relatively small.
It is a three-dimensional structure.
Substrates bind to the active site by multiple weak interactions (and
sometimes covalent).
Active sites are clefts or crevices. Polar residues are often found in active
sites, to create a specific electrostatic environment.
Induced Fit Hypothesis: Active sites change conformation when bound, to
accommodate the substrate.
Active sites have specificity:
Trypsin is specific for Lys and Arg.
Chymotrypsin is very similar to trypsin except it has different active
sites. It binds to hydrophobic aromatic residues in its active site.
Elastase, also similar, recognizes small residues like Gly and Ala.
Collagenase: Contains two Zinc atoms in its active site: one for catalysis,
and one that helps fold the protein.
MICHAELIS-MENTON KINETICS:
When the concentration of substrate, [S], is very high compared to enzyme,
the enzyme will be saturated with substrate, and the rate of rxn will depend
only on the amount of enzyme present -- not substrate.
vmax = The maximum rate possible, under ideal circumstances, when
substrate concentration is high and enzyme concentration is relatively low.
Given steady state, the rate, vmax = k3[ES] = [rate of formation of
product] x [concentration of ES-Complex]
Four Assumptions of the Michaelis-Menton Model:
A complex between E and S, ES, is formed.
The concentration of S is much larger than E.
The degradation of Product back to the ES-Complex is ignored.
A steady-state concentration of the ES-Complex is established during
measurement.
The steady-state forms practically instantaneously.
Steady state means that the concentration of ES remains
constant, i.e. the rate at which the reaction occurs is uniform.
Total Enzyme, Et, under the steady state = [ES] + [Eunbound]. Some of the
enzyme remains unbound in the steady state.
V0 = initial rate
Vmax = Maximum rate in steady state
Km = The concentration of substrate, [S], at which the current rate, (V0), is
equal to half of the maximum rate, Vmax.
Once Km is known, it is possible to calculate the fraction of sites filled.
Lineweaver-Burk Plot: A plot of the reciprocal of rate -vs- reciprocal of
substrate concentration. It is linear.
IRREVERSIBLE INHIBITION: An inhibitor that covalently and permanently alters
an enzyme, rendering it dysfunctional.
Diisopropyl Phosphoro Fluoridate (DFP): Is a SERPIN -- Serine Proteinase
Inhibitor.
Iodoacetamide irreversibly inactivates cysteine residues by alkylating
them.
Aspirin is a Non-Steroidal Anti-Inflammatory Drug (NSAID). It is an
irreversible inhibitor of Prostaglandin-H Synthetase, the enzyme that makes
prostaglandins. Aspirin is thus an anti-inflammatory drug.
Prostaglandin has cyclooxygenase activity: Arachidonic Acid ------>
PG2
Prostaglandin has hydroperoxidase activity: PG2 ------> PGH2.
Penicillin: Irreversibly inhibits the synthesis of peptidoglycans in bacterial
cell walls.
It inhibits glycopeptide transpeptidase, which cross-links poly-Gly
with D-Ala. It imitates the Ala-D-Ala.
Transition-State Analog: It has a four-member lactam ring, highly
strained, and it imitates the transition state of Ala-D-Ala in the
reaction above.
REVERSIBLE INHIBITORS:
Competitive Inhibitor: The inhibitor resembles (mimics) the substrate and
thus competes directly with the substrate for the same active site.
Competitive Inhibitions increases km -- a higher substrate
concentration is required to achieve the same vmax. Hence adding
more substrate can alleviate the effects of the inhibition.
Lineweaver-Burk plot: the slope increases.
2,3-bisphosphoglycerate mimics 1,3-bisphosphoglycerate. Malonate
mimics succinate.
Non-Competitive Inhibitor: Allosteric Inhibitor. The inhibitor hooks to a
different site on the enzyme, which can temporarily render it dysfunctional.
No matter how much substrate you add, you can't dilute the effects.
Hence vmax is decreased. km does not change!
alpha1 Proteinase Inhibitors: A mutation in alpha1-antitrypsin (SerineProteinase-Inhibitor family, SERPINs) caused it to mimic caused the protein to
inhibit thrombin instead of trypsin, which led to hemophiliac symptoms! Mutation
of Arg for Met.
The alpha1-Antitrypsin normally inhibits trypsin, neutrophil elastase, and
cathepsin-G.
In its absence emphysema can also form due to the lost inhibition of
neutrophil (i.e. macrophage) elastases in the lungs.
Smoking causes the oxidation of the key Met-358 to methionine sulfoxide,
thus ruining the anti-elastase activity ------> emphysema.
alpha2 Macroglobulin: Molecular Trap: Sterically hinders (traps)
endopeptidases. This enzyme is found in serum in large quantities.
Endopeptidase: Enzymes that cleave proteins only on the inside.
Exopeptidase: Enzymes that cleave proteins only on the outside (terminal)
part.
Aminopeptidase: Exopeptidase that cleaves at the amino terminus.
Carboxypeptidase: Exopeptidase that cleaves at the carboxy
terminus.
Molecule has four subunits, two of which are disulfide linked. There is a bait
region which almost all endopeptidases recognize. The peptidases bite the
bait, and the macroglobulin changes conformation so as to physically
enclose ("trap") the molecule.
The enzyme is still catalytically active! It just sterically hinders the
peptidases so that peptides can't reach them. It does inhibit them.
Physiologic Purpose: alpha2-Macroglobulin traps proteolytic enzymes that
are released during edema.
Principles of Enzyme Assay:
The initial rate of reaction should be measured only.
Zero-order: Large excess of substrate.
vmax can still be reached to 90%, even when using a high
concentration of substrate. High substrate conc should be used so
that rate is proportional to enzyme concentration only.
Spectrophotometry: Can measure trypsin by visualizing its cleavage
products with p-nitroanaline (410 nm).
Collagenase: Collagenase cuts collagen at one locale in order to denature all
three sub-chains.
Has important roles in physiology roles and pathology.
Physiological roles:
Embryo implantation in uterus. Collagenase eats its way through
endometrium.
Pathology:
Arthritis: Collagen II is found in normal joints. It may start to
deteriorate, via too much collagenase activity.
Rheumatoid Arthritis: Synovial membranes in the joints
proliferate around the collagen (II), and then start to degrade.
Osteoarthritis: Auto-immune disease causing bone breakdown
(self-digestion by macrophages).
Metastasis: Collagenase must eat through ECM for cancer cells
to spread.
Detecting Collagenase activity: Difficult. It recognizes a stretch of residues.
Some flourescent techniques have been developed.
Measuring a Reaction by Coupling it to another Reaction:
Lactate Dehydrogenase (LDH) can be measured by coupling it to
hydrazine.
Reaction: Pyruvate ------> Lactate. It normally tends toward lactate
strongly.
In the presence of hydrazine, it removes pyruvate, pulling the reaction
in that direction. (keq for that reaction is strongly in the other
direction).
So use hydrazine to measure the reverse reaction (i.e. the formation
of pyruvate).
Another example of coupling: Phosphoenol-pyruvate + ADP ------>
Pyruvate + ATP.
This rxn normally tens toward the PEP. But use NADH to drive it the
other direction.
NADH depletion can then be measured photometrically.
UNITS: Amount of product produces / unit time. A measurement of enzyme
activity.
SPECIFIC ACTIVITY: Units / mg protein. A measurement of the reactivity of a
protein, or the relative rate of reaction for an enzyme. It is total enzyme activity /
total protein.
Specific Activity will increase as the enzyme becomes purified.
Can always measure specific activity as (Volume)(Units / mL) / (Total
protein) = (Units / mg)
ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA): A way of measuring the
amount of enzyme present, not its activity.
Procedure:
Attach an antibody specific to the protein being measured.
Add the unpurified sample. The antibody will attach to the protein
being measured.
Add a second antibody specific for the enzyme (at a different antigen
site). The second enzyme is already coupled to an indicator. This will
create a solid platform of hooked enzyme.
Add the substrate and measure the product, usually photometrically.
NEOEPITOPE ANTIBODY ASSAY: It measures newly regulated antibody binding
sites. Can be sued to measure the cleavage products of collagenase.
Mechanisms of Enzyme Catalysis:
Proximity Effect: Reaction rate is increased when reactants are closer
together, i.e. there is a higher probability of collision. Enzymes can act by
bringing the reactants closer together.
As the free rotation of the reactants decreases, reaction rates
increase, as the reactants can be brought closer together (in right
orientation) with more ease. This is also the proximity effect.
Enzymes decrease the possible loss of entropy, which decreases the
activation energy.
General Acid-Base Catalysis: Enzymes act as acids and bases.
His residues often catalyze hydrolysis of ester ------> acid + alcohol.
It can do it at neutral pH.
Covalent Catalysis.
Nucleophilic groups can attack to form nucleophilic intermediates (such as
acyl intermediates)
Coenzymes can participate in the reaction stoichiometrically (such as
NADH).
Enzymes can lower activation energy by inducing a strain on the substrate,
making it more energetically favorable to go into the transition state.
Protease: Describes both endopeptidases and exopeptidases.
Proteinases: Cleave proteins and are thus only endopeptidases.
Serine Proteases: Enzyme that use Ser as a nucleophile in the active site, in
order to cleave proteins at specific residues.
DFP is a good agent to use to recognize them. It is specific to Ser
proteases.
Examples:
Trypsin
Chymotrypsin
Neutrophil Elastase
Pancreatic Elastase
Thrombin -- very similar to trypsin
TPA: Tissue Plasminogenic Activator
Cysteine Proteases: Similar to Ser proteases, except Cysteine does the cleaving
at the active site. Cysteine has sulfur (thiol) -vs- Serine's oxygen (alkoxide) as
the nucleophile.
Inhibited (and identified) by iodoacetate.
Catalysis proceeds through a thiol-ester intermediate.
Examples:
Lysosomal Enzymes! Cathepsins B, H, L, S.
Papain, from papaya
Interleukin 1b converting enzyme
Aspartic Proteases: Have two Asp residues as the active cleavers.
No good specific inhibitors as above, but pepstatin is a good competitive
inhibitor.
Optimal pH for the enzyme is often acidic.
Examples:
Pepsin
Cathepsin D
Renin, Rennin
HIV Proteinase
Metalloproteases: Often contain Zn+2 in the active site.
Examples:
Collagenase is a matrix metalloproteinase.
Carboxypeptidase A and B
Thermolysin -- a bacterial enzyme that has three residues (His, His,
Glu) that "hold" Zn in place.
The Zn+2 attacks the carbonyl oxygen, making the
electropositive center. Water then attacks the carbonyl carbon.
A nice tetrahedral intermediate is formed once again.
His acts as an acid in the reaction -- relinquishes a proton to the
tetrahedral intermediate.
CATALYTIC MECHANISM OF CHYMOTRYPSIN: A Serine Protease
Ser-195: It was discovered that it was a two-step mechanism because of
the burst-assay. First the rate went fast and then it leveled off.
They tested it with p-nitrophenol. Two-step process was revealed.
Step-1 = release of the p-nitrophenol group.
Step-2: Hydrolysis of the acetate to form the acetyl enzyme
intermediate.
His-57: The catalytic role os His-57 was discovered by affinity-labeling.
TPCK, Tosyl phenylalanine chloromethyl ketone, specifically binds to
the His residue.
The phenylalanine goes into the hydrophobic chymotrypsin pocket,
and it turns out that the chloromethyl part is bound to a histidine.
The reaction is highly stereospecific. It won't work with
D-chymotrypsin.
Catalytic Triad: Ser-195, His-57, Asp-102. All three participate in the
cleavage reaction.
His accept the proton from Ser-OH, making Ser-O- a nucleophilic
alkoxide moiety.
Asp- stabilizes the positive charge created on His+.
A tetrahedral transition state is formed.
An oxoanion pocket is formed in the intermediate, with Ser-O- residue
central in the pocket.
Cleavage occurs at the Carbonyl-Nitrogen bond, and the C-terminus
peptide is released into solution.
There is a charge-relay network in the catalytic triad, a stabilization of
charge.
Cofactors and Coenzymes:
Apoenzyme: Inactive form of enzyme without the cofactor present.
Holoenzyme: Active enzyme with the cofactor present.
NAD+/NADH NADP+/NADPH: Modified forms of the vitamin niacin.
The functional group is nicotinamide... which is oxidized and reduced.
FMN/FMNH FAD/FADH2: Flavin Adenine Dinucleotide, from riboflavin,
vitamin B2.
Vitamin B6 (pyridoxine): Works as a cofactor for transaminases in protein
metabolism.
Transfers an amino group from an amino acid to a keto acid.
The cofactor is covalently attached to the enzyme. It combines with a
Lys residue in the transaminase to form a Schiff base.
Vitamin B12: Cobalamin: Main part consists of a corrin ring, which remains
attached to four nitrogen atoms (similar to a heme group).
At the sixth coordination position, the most reduced state can accept
the 5' deoxy adenosyl group. Otherwise it can't. That depends on the
oxidation state of cobalt.
Lack of Vit-B12 leads to a problem in making methionine, in amino
acid metabolism.
Tetrahydrofolate is needed for production of purines and pyrimidines.
THF synthesis requires Vitamin B-12. Hence, in the absence of B-12,
purines and pyrimidines (i.e. new DNA) can't be reduced, so cells
don't proliferate.
Result = anemia, lack of proliferation of red blood cells. CONC:
Vit-B12 deficiency causes anemia.
RIBOZYMES: RNA-Enzymes
It is self-splicing RNA. It can act as a catalyst for its own splicing.
L19 Ribozyme can act as an endonuclease. It has the pentacytidine and
RRRRR mechanism.
Blood Coagulation:
Intrinsic Pathway: Only very small amounts of the early materials are
present (or need be present).
Damaged surface stimulates Kininogen and Kallikrein.
XII ------> XIIa
XI ------> XIa
IX ------> IXa (Ca+2 required)
With help of VIIa, IXa activates X ------> Xa
Extrinsic Pathway:
Trauma starts it (Damaged tissue)
VII ------> VIIa
It activates X ------> Xa
Common Pathway:
V ------> Va (Ca+2 required)
Prothrombin ------> Thrombin (Ca+2 required)
Pro-Thrombin has gamma-Carboxyglutamic Acid, a
post-translational modification of Glu residues.
Vitamin-K is required to make that modification. Vitamin-K is
important in other factors as well. Vitamin-K deficiency leads to
hemorrhaging and hemophiliac symptoms.
Dicumarol is an antagonist to Vitamin-K. Drugs containing
dicumarol or dicumarol analogs are used in anti-coagulant
therapy.
Fibrinogen ------> Fibrin
Two A fibrinopeptides and 2 B fibrinopeptides are removed.
Fibrin (alphabetagamma)2 is left.
Factor XIII ------> XIIIa cross-links with the fibrin.
Cross-links Glu and Lys residues.
Thrombolysis: Regulation and reversal of clotting
Antithrombin III most strongly inhibits thrombin.
Heparin binds to Antithrombin III and allosterically enhances its
effect on Thrombin.
Thrombin self-regulates by activating Protein C, which digests factors Va
and VIIIa.
Normal Clot Lysin:
Tissue Plasminogen Activator (TPA) activates Plasminogen ------>
Plasmin.
TPA is released from endothelial cell only when there is local
injury.
When a person gets older, TPA doesn't work as well, and
clot-buildup (atherosclerosis) results.
Enzyme Regulation in the Duodenum:
Zymogen: Inactive, uncleaved precursor to an active enzyme. The zymogen
is usually activated by proteolysis.
Chymotrypsinogen is activated by Trypsin.
First forms PI-Chymotrypsin, which is itself active.
Then PI-Chymotrypsin self-cleaves to form the still-active alpha-
Chymotrypsin, which contains three chains that are disulfide linked.
Trypsinogen is changed to Trypsin via enteropeptidase in the duodenum.
Trypsin also activates proelastase and procarboxypeptidases.
Pepsinogen auto-cleaves itself to convert to Pepsin, in an acidic
environment in the stomach. The propeptide is more basic while the active
enzyme is more acidic.
Reversible Covalent Modifications:
Glycogen Phosphorylase: In skeletal muscle.
Phosphorylase A, R (relaxed, active) form, is the most active form. It
is phosphorylated.
Phosphorylase B, T (taut, inactive) form, is the least active form.
Phosphorylase Kinase catalyzes the transformation of Phosphorylase
B ------> Phosphorylase A.
Isozymes: Enzymes that have different chemical and physical properties, but that
catalyze the same reaction. They may have different kinetics.
Lactate Dehydrogenase (LDH) has the H (heart) and M (muscle) forms.
There are four subchains and five combinations of them, ranging from H 5 to
M5 .
LDH-1 (pure H) is found in heart. It favors pyruvate over lactate, so
that pyruvate isn't used for anaerobic respiration when it needs to be
used for aerobic. The km for pyruvate is low.
LDH-5 (pure muscle M) is found in skeletal muscle. It favors formation
of lactate, to reoxidize NADH in skeletal muscle. That is necessary, as
muscles work in bursts.
CORI CYCLE: Glycolysis and Gluconeogenesis. Same enzymes used in both
places.
Skeletal Muscle: Glucose ------> Pyruvate ------> Lactate.
Liver: Lactate ------> Pyruvate ------> Glucose.
ASPARTATE TRANSCARBAMYLASE (ATCase):
It is an enzyme used to form CTP (part of nucleic acid synthesis). The
presence of CTP inhibits its via allosteric negative feedback.
The presence of ATP allosterically stimulates its (increases rate). So, when
ATP is around more of the stuff is made to make more nucleic acid for cell
division.
The curve is sigmoidal, indicating cooperative type interactions, where the
activation of some encourages even more.
The substrate binding changes the 4 conformation.
Mercurial compounds like p-hydroxymercuribenzoate, desensitize the
enzyme. It still has activity at the active site, but the regulatory sites no
longer work. No more positive or negative allosteric regulation.
Concerted Model: Allosteric Effects
There are two forms, R (relaxed, active) and T (taut, inactive).
There is virtually no intermediate. The substrate binds the T-form, which
causes practically instantaneous conversion to the R-form, allowing more
binding (or complete binding) of substrate.
Sequential Model: Has a combination form possible (intermediate) of half T-form
and half R-form.
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Copyright 1999, Scott Goodman, all rights reserved