Exam 1 Flashcards
(267 cards)
1
Q
A
ethyl
2
Q
A
phenyl
3
Q
A
carbonyl (aldehyde)
4
Q
A
carbonyl (ketone)
5
Q
A
carboxylate
6
Q
A
hydroxyl (alcohol)
7
Q
A
ether
8
Q
A
ester
9
Q
A
acetyl
10
Q
A
anhydride
11
Q
A
amino (protonated)
12
Q
A
amido
13
Q
A
sulfhydryl
14
Q
A
disulfide
15
Q
A
thioester
16
Q
A
phosphoryl
17
Q
R/S configuration rules
A
- Compare the atomic number (Z) of the atoms directly attached to the stereocenter; the group having the atom of higher atomic number receives higher priority.
- If there is a tie, consider the atoms at distance 2 from the stereocenter—compare the atoms bonded to the one directly attached to the stereocenter; at the earliest difference, the group containing the atom of higher atomic number receives higher priority.
Br > Cl > S > –OCH3 > –OH > –NH2 > –COOH > –CHO > –CH2OH > –CH3 > –H
18
Q
water hydrogen bonding
A
- Water molecules attracted to one another due to partial charge differences between O and H - Hydrogen bond
- strong noncovalent attraction
- Almost tetrahedral shape allows for water molecules to H-bond with 4 other water molecules -> ice
19
Q
hydrogen bonds
A
readily form between any electronegative atom (the hydrogen acceptor) and a hydrogen atom covalently bonded to another electronegative atom (the hydrogen donor)
20
Q
water polarity
A
- Due to it’s polarity and ability to hydrogen bond, water can act as a polar solvent to dissolve charged or polar molecules by hydrating them
- Since many biomolecules are either charged or polar they easily dissolve in water (hydrophilic) and are soluble in the aqueous environment of the cell
- Water dissolves salts by weakening the electrostatic interactions between the ions that allow it to form a crystal lattice
- Solvation causes increase in entropy since ions are freed from crystalline structure thus delta G is negative (ΔG= ΔH-TΔS)
21
Q
hydrophobicity
A
- When a non-polar solute is placed in water, the water molecules in the immediate vicinity of the non-polar molecule are constrained in their orientations.
- The result is an overall increase in entropy of the water ΔS > 0 since water becomes less restricted upon micelle/cluster formation.
*
22
Q
ordered waters in binding interactions
A
Individually these are small energies, but collectively they make large contributions to biochemical processes.
23
Q
acid/base property of water
A
water can ionize
24
Q
ionization of water
A
In non pure water solutions, [H+] and [OH-] will change but together still must equal a Kw of 1 x 10-14 M2
Kw is basis for pH scale which tells concentration of H+ and therefore
OH- as well
25
pka is related to...
```
the degree of acid dissociation
Strong acids (e.g. HCl, H2SO4 and HNO3) & strong bases (e.g. NaOH and KOH) ionize completely at dilute concentrations.
```
Weak acids and bases, which do not completely ionize in solution, are more biologically relevant!!
26
strong acid -\>
large Ka -\> small pKa
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small pka -\>
large Ka -\> strong acid
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weak acid -\>
small Ka -\> large pKa
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large pKa -\>
small Ka -\> weak acid
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buffer
* aqueous systems that resist changes in pH due to small amount of acid or base
* Decrease in one component balanced by increase of another to maintain equilibrium of two reactions ( ionization of water and the weak base)
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titration curve
buffers are maximally effective at...
Buffering region is where...
* buffers are maximally effective at its pKA value
* Buffering region is where the pH changes the least upon addition of a hydrogen ion or a hydroxide ion
32
HH equation
describes shape of a titration curve since pH is y
axis
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how HH eq can be rearrange to solve for conjugate acid base ratio
34
when pH\>pKa
acid/base
[A-]/ [HA] ratio is greater than 1 so [A-] \> [HA]
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when pH \< pKa
acid/base
[A-] / [HA] ratio is less than 1 so [A-] \< [HA]
36
amino acid structure
carboxyl group and an amino group attached to the α-carbon
differ in R group
37
R group can effect:
* Hydrophobicity/hydrophilicity
* Polarity
* Charge
* Isoelectric point
* Acidity/basicity
* Steric bulk
* Spectroscopic activity
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amino acids are in ___ configuration
L
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D,L method
40
nonpolar aliphatic R groups
glycine, alanine, proline, valine, leucine, isoleucine, methionine
L, I, M, P, V, A, G
* Nonpolar can stabilize protein structure via hydrophobic effect
* Tend to cluster together within protein
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polar, uncharged R group
serine, threonine, cysteine, asparagine, glutamine
S, C, Q, N, T
* Polar is very hydrophilic
* can form H- bonds with water
* more soluble in water
42
positively charged R groups
lysine, arginine, histidine
H, R, K
* all are basic
* histidine can also have a charge when double-bonded N of ring protonated at pH 7
43
negatively charged R groups
aspartate, glutamate
E, D
* Great proton acceptors
44
aromatic R groups
phenylalanine, tyrosine, tryptophan
F, Y, W
* good for examining protein concentration
* Relatively nonpolar, contribute to hydrophobic effect
* Hydroxyl group of tyrosine can form hydrogen bonds
* Tyrosine and tryptophan are significantly more polar than phenylalanine
* Aromatics have characteristic absorbances near 280nm
45
type of group
glycine Gly G
nonpolar, aliphatic R group
46
type of group
nonpolar, aliphatic R group
alanine ala A
47
type of group
nonpolar, aliphatic R groups
proline pro P
48
type of group
nonpolar, aliphatic
valine val V
49
type of group
nonpolar aliphatic
leucine leu L
50
type of group
nonpolar aliphatic
isoleucine Ile I
51
type of group
nonpolar aliphatic
methionine met M
52
type of group
polar, uncharged
serine ser S
53
type of group
polar uncharged
threonine thr T
54
type of group
polar uncharged
cysteine cys C
55
type of group
polar uncharged
asparagine asn N
56
type of group
polar uncharged
glutamine gln Q
57
type of group
positively charged
lysine lys K
58
type of group
positively charged
arginine arg R
59
type of group
positively charged
histidine his H
60
type of group
negatively charged
aspartate asp D
61
type of group
negatively charged
glutamate glu E
62
type of group
aromatic
phenylalanine phe F
63
type of group
aromatic
tyrosine tyr Y
64
type of group
aromatic
trytophan trp W
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disulfide bond formation
* Disulfide bonds can link multiple segments of a protein and stabilize protein structure via covalent bond formation
66
Post-translational Disulfide bond formation:
modifications (PTM’S)
PTM’s can turn protein function on or off, target it for degradation or for recognition by another protein
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zwitterions and amino acids
* nonionic form of amino acids does not occur in significant amounts in aqueous solution
* @ neutral pH - zwitterionic (dipolar hybrid ion) form predominates
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amphoteric
zwitterion can act as an acid (proton donor) or base (proton acceptor)
69
titration curve
parts
* First stage of titration: COOH group of glycine loses its proton
* Midpoint of this stage: equimolar concentrations of proton donor and proton-acceptor species are present
* Point of inflection: pH is equal to the pKa of the protonated group being titrated
* Another point of inflection: removal of first proton is complete and removal of the second begins
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HH equation ratio
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isolectric point
* An amino acid is isoelectric when the overall charge of the molecule is 0.
* The pI value or isoelectric point for an amino acid with nonionizable R-groups is determined by averaging the pK1 and pK2 value.
72
At pH \> pI molecule will be
negative
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At pH \< pI molecule will be
positive
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ionizable groups
His, Lys, Arg, Asp, Glu, Tyr, Cys
H, K, R, D, E, Y, C
K, D, H, E, C, R, Y,
if ionizable then has pKr
75
Negative index value (ΔG\<0) ⇒
favorable, i.e. hydrophilic
76
Positive index value (ΔG\>0, ⇒
unfavorable, i.e hydrophobic
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G
gly
glycine
78
A
alanine ala
79
P
proline pro
80
V
valine val
81
L
leucine leu
82
I
isoleucine Ile
83
M
methionine met
84
S
serine ser
85
T
threonine thr
86
C
cysteine cys
87
N
asparagine asn
88
Q
glutamine gln
89
K
lysine lys
90
R
arginine arg
91
H
histidine his
92
D
aspartate asp
93
E
glutamate glu
94
F
phenylalanine phe
95
Y
tyrosine tyr
96
W
trytophan trp
97
gly
G
glycine
98
ala
alanine A
99
pro
proline P
100
val
valine V
101
leu
leucine L
102
Ile
isoleucine I
103
met
methionine M
104
ser
serine S
105
thr
threonine T
106
cys
cysteine C
107
asn
asparagine N
108
gln
glutamine Q
109
lys
lysine K
110
arg
arginine R
111
his
histidine H
112
asp
aspartate D
113
glu
glutamate E
114
phe
phenylalanine F
115
tyr
tyrosine Y
116
trp
trytophan W
117
acid/base character of a peptide/protein can be predicted based on its...
free α-amino and α-carboxyl terminal groups as well as the nature and number of it ionizable R groups
118
Calculating pI of a protein
1. Identify ionizable groups
2. Find charge at acidic, neutral and basic conditions ( ex pH: 3, 8, 11)
3. Average pK’s immediately before/ after zero charge
119
pH \> pKa
molecule deprotonated
120
pH \< pKa
molecule porotonated
121
drawing peptides
* Trans stereochemistry
* L/S configuration except Cys (L/R)
* N to C terminus orientation
122
subunit
Noncovalently associated polypeptides that can form a structure by itself
123
oligomeric
When a small number of subunits associate to yield the functional protein complex
124
multimeric
Proteins with many associated subunits
125
Proteins vary widely based on:
1. Number of residues in the polypeptide(s) making up the protein
* Naturally occurring proteins vary in length from two to many thousands of amino acid residues
* The vast majority of proteins contain fewer than 2,000 residues
2. Often these residues are part of a single, covalently linked amino acid chain, but some proteins utilize 2 or more polypeptide chains to form the functional protein.
3. Amino acid composition and sequence of the polypeptide chains
* Amino acids are not all equally represented in proteins
* The sequential order of the amino acids varies
4. The incorporation (or not) of prosthetic group(s)
* The non-amino acid component of a conjugated protein is referred to as the prosthetic group
* Many proteins contain only amino acid residues (simple proteins)
* Other proteins contain permanently associated chemical components in addition to their amino acids (conjugated proteins)
126
covalent peptide bonds
dictates conformation
* The alpha-carbons of adjacent residues are separated by three covalent bonds
* X-ray diffraction studies of polypeptides showed that the peptide bond (C-N, 2) is shorter than the N-C⍺, 3 bond.
* Due to the partial double bond character, the peptide C-N bond cannot rotate freely – limiting the possible conformations of the polypeptide chain.
127
Ramachandran plot
shows ‘allowed’ Φ/Ψ angles
128
α helix
secondary structures
* H-bonding between the N—H of 1 residue and C=O of 4
* residue away stabilizes structure (parallel to axis of helix)
* Right handed twist with side chains sticking out
* has overall helix dipole
* peptide bond has strong dipole moment
* α-helix has a large macroscopic dipole moment that is + at amino terminus and – at carboxyl terminus
* Negatively charged residues often occur near the positive end of the helix dipole thus aiding in stability
129
Five constraints affecting the stability of an α- helix
1. The intrinsic propensity of an amino acid residue to form an α-helix
2. The interactions between R groups, particularly those spaced 3 (or 4) residues apart
3. The bulkiness of adjacent R groups
4. The occurrence of Pro and Gly residues
5. Interactions between amino acid residues at the ends of the helical segment and the electric dipole inherent to the α helix
130
β strands
secondary structure
* the backbone of the polypeptide chain is extended into a zigzag rather than a helical structure
* R groups of a β conformations extend above and below the strand in an alternating manner perpendicular to the backbone carbonyl oxygens and amine hydrogens
131
Parallel β sheets
* arrangement of several segments side by side, all of which are in the β conformation
* The zigzag strands can be arranged side by side to form a pleated sheet (β sheet) with adjacent polypeptide chains arranged in parallel or antiparallel orientations.
* Gly and Pro residues often occur in β turns
* Gly - because it is small and flexible
* Pro - because peptide bonds involving the imino nitrogen of proline readily assume the cis configuration
132
Circular dichroism spectroscopy
measures the difference in absorption of left-handed and right-handed plane-polarized light resulting from structural asymmetry in a molecule
133
Factors favoring unfolding:
* loss of entropy of the polypeptide chain-environmental changes
* loss of many hydrogen bonding interactions of the polypeptide chain to water
134
Factors favoring folding:
* hydrophobic interactions resulting from the shielding of hydrophobic residues from water
* van der waal interactions
* Hydrogen bonding
* ionic interactions between nearby charged groups on the polypeptide
* disulfide (covalent bond) formation
135
Tertiary and quaternary structures can be:
fibrous or globular
136
Fibrous + ex
* Polypeptide chains arranged in long insoluble strands or sheets
* Usually one type of secondary structure, with relatively simple tertiary structure
* Provide support, shape, and external protection
* Insoluble in water → high concentration of hydrophobic amino acid residues
* ex: α-keratins, collagen, silk
137
globular + ex
* polypeptide chains folded into spherical or globular shape
* contain several types of secondary structure
* 3D structure - assemblage of polypeptide segments in the α- helix and β conformations, linked by connecting segments
* Ex: enzymes, transport proteins, motor proteins, regulatory proteins, immunoglobulins, and proteins with many other functions
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motif
(also known as a fold or supersecondary structure) is a recognizable folding pattern involving two or more elements of secondary structure and the connections between them
139
keratin
fibrous, built from coiled coils
* Individual polypeptide strands arranged in a right handed helix
* Disulfide bonds link keratin α-helices
* keratin strength due to # of cysteines
* Tertiary structure is coiled coil motif stacked into protofibrils
* Coiled-coiled structure are two right handed α-helix polypeptides wrapped left-handed
* Quaternary structure is intermediate filaments that make up hair strand
140
silk
fibrous, made up of β-sheets
* Spider silk made up of the protein fibroin that is made up of beta strands
* tertiary structure is many anti parallel beta strands stacked on one another
* H-bonds stabilize strands, van der waals interactions stabilize sheets
* Structure allows flexibility but not stretching
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globular proteins
* Globular proteins have a great variety in tertiary structures.
* The three-dimensional structure of globular proteins can exhibit differing proportions of α- helical and β- conformational character.
* These secondary structural components can combine in more complex patterns.
142
Models of folding: Hierarchical/stepwise
1. Local secondary structures form (ionic interactions can be important)
2. Longer range interactions between secondary structures form
3. Process continues until complete domains form and the entire polypeptide is folded
143
Models of folding: Molten Globule (partially folded)
Spontaneous collapse of the polypeptide into a compact state (molten globule) which is mainly driven by hydrophobic interactions
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denaturation
* Changes in a protein environment can cause loss of native structure.
* Denaturation is a loss of 3D structure sufficient to cause loss of function
* The abrupt loss of structure suggests unfolding is a cooperative process
145
Proteins can be denatured by:
* heat - complex effects on many weak interactions in a protein (primarily on the hydrogen bonds)
* pH extremes - alter the net charge on a protein, causing electrostatic repulsion and the disruption of some hydrogen bonding
* detergents, solutes (such as urea, Guanidinium) - disrupt the hydrophobic aggregation of nonpolar amino acid side chains that produces the stable core of globular proteins
* urea disrupts hydrogen bonds
146
Renaturation
the process by which a denatured protein regains its native structure and biological activity when returned to conditions in which the native protein is stable
147
anifsen experiment
\*First evidence that the primary amino acid sequence provides all the necessary information to fold the chain into its native, 3-D structure, aka the “Thermodynamic Hypothesis”- Done before structures were available!
148
non-covalent binding
Some protein functions interactions are reversible
(reversible interactions)
149
ligands
molecules (small molecules, proteins, etc) that reversibly bind a protein
150
binding site
Ligands bind at a site on the protein
151
Ligand Binding Models
lock and key, induced fit
152
lock and key model
* Binding site complements the ligand in size, shape, charge, or hydrophobic/hydrophilic, character
* must be perfect fit
153
induced fit model
* Ligand binding causes a structural change slightly different from the native structure in the absence of ligand.
* Ligand or protein can change its conformation
154
Ka
association constant
155
Kd
dissociation constant
corresponds to the ligand concentration at which 50% of the binding sites are occupied
156
higher Ka =
low Kd = higher affinity of ligand for protein
157
lower Ka =
high Kd = lower affinity of ligand for protein
158
low Kd =
high affinity
159
high Kd =
low affinnity
160
Problems with O2 binding
* Protein side chains lack affinity for O2
* Some transition metals bind O2 well but would generate free radicals if free in solution
* Organometallic compounds such as heme are more suitable, but Fe2+ in free heme could be oxidized to Fe3+ which cannot bind O2
161
oxygen binding solution
globulins
* Globins use heme permanently bound to Fe2+, to bind oxygen
* Heme is buried in core of protein to avoid Fe2+ to Fe3+ conversion!
* Oxygen coordinated to iron causes small structural changes
* this happens in both myoglobin and hemoglobin
162
Oxygen binding by myoglobin is...
hyperbolic
163
Myoglobin great for
O2 storage but not the release of it for when cells need more O2
164
hemoglobin binding
* Hemoglobin is Allosteric - Binding of oxygen to hemoglobin induces structural changes
* Hemoglobin undergoes a transition from a low- affinity state (the T state) to a high-affinity state (the R state) as more O2 molecules are bound → hemoglobin has a hybrid S-shaped, or sigmoid, binding curve for oxygen
165
hemoglobin T state
“tense”
* Stabilized by structural ion pairs most of which lie in the α1β2 (and α2β1) interface
* weaker affinity for O2
* When oxygen is absent, T state is more stable
* Stabilized by greater number of ion pairs
166
hemoglobin R state
“relaxed”
* Stabilized by O2
* Stronger affinity for O2
* O2 binding triggers a T → R conformational change
* αβ subunit pairs slide past each other and rotate narrowing the pocket between the β subunits.
167
allostery
communication between two sites on a protein
Ligands bound can be the same (homotropic) or different (heterotropic)
168
cooperativity is a form of...
allostery
169
positive cooperativity
when binding promotes other ligands to bind to a protein in such a way that the mutually reinforce each of their binding affinities
170
negative cooperativity
when binding inhibits other sites
171
Hemoglobin binding of oxygen is...
sigmoidal
* Hemoglobin affinity for oxygen changes with pO2 environment
* Characteristic due to positive cooperative binding
172
p50
pressure where ½ of protein bound by ligand
173
Sequential model-in terms of hemoglobin
* Ligand binding can induce a change of conformation in an individual subunit
* More potential intermediate states than in concerted model
* O2 binding changes conformation one subunit at a time
* One hemoglobin can have subunits that is more like R or more like T conformation
174
MWC/concerted model-in terms of hemoglobin
* Assumes that the subunits of a cooperatively binding protein are functionally identical
* O must completely bind to all subunits in T state to change to R and all O must release to convert to T state
* T and R state in equilibrium with O2 biased toward R state
* No protein has individual subunits in different conformations → the two conformations are in equilibrium
175
Hills coefficient (nH)
represents cooperativity and is slope of Hill equation
176
nH = 1
ligand binding is not cooperative
can arise even in a multisubunit protein if the subunits do not communicate
177
nH \> 1
positive cooperativity
178
nH \< 1
negative cooperativity
the binding of one molecule of ligand impedes the binding of others
179
2,3-bisphosphoglycerate (BPG)
* ligand that is naturally found bound to hemoglobin in red blood cells
* [BPG] low enough to not effect O2 binding in lungs
180
Regulation of oxygen binding
BPG
* One BPG bound to the hemoglobin tetramer lowers affinity for oxygen by stabilizing the T state
* O2 binding causes collapse of BPG binding pocket inn R state
* important for changes to O2 levels ex: high altitudes vs sea level, active repairing muscles
181
Regulation of oxygen binding
CO
* CO has similar size and shape to O2; it can fit to the same binding site.
* CO binds heme over 20,000 times better than O2 because the carbon in CO has a filled lone electron pair that can be donated to vacant d-orbitals on the Fe2+.
* CO is highly toxic, as it competes with oxygen. It blocks the function of myoglobin, hemoglobin, and mitochondrial cytochromes that are involved in oxidative phosphorylation.
* The differences in affinity are partially explained by steric hindrance from the protein.
182
Bohr effect
pH regulation of oxygen binding
Binding of H+ and CO2 is inversely related to the binding of oxygen
* Hemoglobin also carries H+ and CO2, from the tissues to the lungs and the kidneys
* Low pH and high CO2 concentration of peripheral tissues → affinity of hemoglobin for oxygen decreases as H+ and CO2 are bound → O2 is released to the tissues
* In capillaries of lungs, as CO2 is excreted and the blood pH rises → affinity of hemoglobin for O2 increases → protein binds more O2 for transport to peripheral tissues
* The binding of H+ and CO2 decreases the binding of O2 thus **shifting plot to right**
* CO2 modifies the N-termini and lowers the pH and high H+ increases protonation of His HC3 stabilizes T state conformation
183
immune system & molecular recognition
* Immunoglobulin G (IgG) has 4 polypeptide chains: two heavy chains and two light chains linked by noncovalent interactions and disulfide bonds
* Specificity is determined by the residues in the variable region of its heavy and light chain (VH and VL)
* Structure stabilized by covalent disulfide bonds
* Follows induce fit model of binding
184
Two common ways to detect only molecule of interest
both involve use of primary antibody to recognize molecule of interest and then a secondary antibody to recognize primary antibody
185
myosin and actin
* ATP binds N terminus head of myosin
* Hundreds of myosin molecules are arranged with their fibrous tails of myosin associating to form thick filaments
* Molecules of monomeric actin (G-actin) associate to form thin
filaments (F-actin)
* Each actin monomer in the thin filament can bind specifically to one myosin head group.
* Provides track for myosin
186
myosinn movement mechanism
ATP hydrolysis provides energy for myosin movement
187
muscle contraction
Regulation prevents continuous muscle contraction
* availability of myosin-binding sites regulated by troponin and tropomyosin to avoid continuous muscle contraction.
* Nerve impulses triggers release of Ca2+
* Ca2+ binds Troponin C subunit that shifts conformation of troponin and tropomyosin to allow myoglobin binding
188
enzymes
* reaction catalysts of biological systems:
* high specificity for their substrates
* greatly accelerate chemical reactions
* function in aqueous solution and under relatively mild conditions of temperature and pH
* often use cofactors/coenzymes
* native protein structure is essential to catalytic activity
* Enzymes broken down to their component amino acids or denatured lose catalytic activity
189
enzyme active site
* three-dimensional cleft or crevice created by amino acids from different parts of the primary structure
* active site constitutes a small portion of the enzyme volume
* create unique microenvironments
* interaction of the enzyme and substrate at the active site involves multiple weak interactions
* Enzyme specificity depends on the molecular architecture at the active site.
190
Reaction coordinate diagrams-No enzyme
* A simple reaction in which substrate (S) is converted to product (P) can be written with the following equilibrium reaction S⇌P
* Energy needed to convert from S to P is shown by reaction coordinate plot
* ΔG‘º determines whether a reaction is spontaneous
* ΔGŧ, determines energy for transition
* Transition state- not a chemical species/intermediate
191
Reaction coordinate diagram-Enzyme
A simple enzymatic reaction can be written: E + S ⇌ ES ⇌EP ⇌E + P
* Enzymes are not consumed!
* Catalysts do not affect reaction equilibria- ΔG‘º unchanged so product formation still favored
* Enzymes lower activation energies
* Cannot make a non-spontaneous reaction occur
192
Noncovalent binding energy
1. Free energy is released from formation of many weak bonds and interactions
2. Weak interactions are optimized in the reaction transition state: enzyme active sites are complementary to the transition state of the enzymatic reaction.
* Enzymes lower activation energy by using energy generated from non covalent interactions!
193
Binding energy in catalysis - lock and key model
* Magnets represent non covalent interactions that occur in a binding site
* No mobility in binding pocket for it to bend
* Strong noncovalent interactions (lock & key fit) makes ES complex very stable thus lower free energy than free S
* Overall increases activation energy to perform transition
* Exact fit of ES interaction site can impede flexibility needed to obtain transition state!
194
Binding energy in catalysis-catalyzed
* Substrate interacts partially with binding pocket so not more stable than free S
* Full non covalent interactions of binding pocket achieved during transition-releasing energy
* ES complex of transition less stable than energy of free S so overall activation energy decreased
* System acquires energy equivalent to the amount needed to lower activation energy from transition interactions (ΔGM)
* overall ΔG‘º unchanged!
195
factor contributing to binding energy in catalysis
1. entropy
2. solvation shell of hydrogen- bonded waters surrounds and helps stabilize biomolecules in solution
3. distortion of the substrates (primarily electron redistribution) that a substrate must undergo in order to react
4. Conformational changes in enzyme upon binding allows for proper alignment of catalytic functional groups on the enzyme
196
factor contributing to binding energy in catalysis
entropy
* High entropy reduces possibility that molecules will react with one another
* Binding energy holds substrates in proper orientation to react (low entropy)
* Low entropy increase chance of productive collisions between molecules in solution
197
factor contributing to binding energy in catalysis
solvation
* solvation shell of hydrogen- bonded waters surrounds and helps stabilize biomolecules in solution
* weak interaction formation between enzyme and substrate displaces those waters
198
Catalytic functional groups contribute to catalysis
Reaction intermediates can be stabilized by the transfer of a proton to or from the substrate or intermediate to form a species that breaks down more readily to products.
199
Specific Acid-Base Catalysis:
Catalysis that uses only the H+ (H3O+) or OH- ions present in water
200
General Acid-Base Catalysis:
* Catalysis in which the proton transfer is mediated by weak acids and bases that is not water such as amino acids
* more crucial in active site where water not present
201
Catalytic mechanisms: chymotrypsin
* Chymotrypsin catalyzes the hydrolytic cleavage of peptide bonds adjacent to aromatic amino acids (F,Y,W)---Cleaves at C-terminus
* Illustrates the principle of transition-state stabilization in catalysis
* Exemplifies the features of covalent and general acid-base catalysis
202
Catalytic mechanisms: chymotrypsin
phase 1
step 1
Phase 1 (Steps 1-4): Peptide bond is cleaved and an ester linkage between the peptide backbone carbonyl and Ser 195 is formed
203
Catalytic mechanisms: chymotrypsin
step 2
Formation of covalent acyl-enzyme intermediate via peptide carbonyl attack by the nucleophilic serine alkoxide
204
Catalytic mechanisms: chymotrypsin
step 3
Collapse of the unstable intermediate breaks the peptide (amide) bond
205
Catalytic mechanisms: chymotrypsin
step 4
Generation of a hydroxide ion for nucleophilic attack of the acyl-enzyme bond
206
Catalytic mechanisms: chymotrypsin
phase 2
step 5
```
Phase 2 (Steps 5-7):
Deacylation with the addition of water to yield the free enzyme
Step 5: Nucleophilic attack of the acyl-enzyme bond
```
207
Catalytic mechanisms: chymotrypsin
step 6
Collapse of the unstable intermediate breaking the acyl-enzyme covalent bond
208
Catalytic mechanisms: chymotrypsin
step 7
Dissociation of the remaining peptide and regeneration of the free enzyme
209
covalent catalysis
* temporary covalent bond is formed between the enzyme and the substrate
* covalent catalyst alters the reaction pathway to lower the activation energy of pathway relative to the uncatalyzed pathway
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metal ion catalysis
* Approximately 30% of known enzymes require one or more metal ions for catalytic activity.
* Ionic interactions between enzyme-bound metals and a substrate can help
* orient the substrate for reactions
* stabilize charged transition states
* Metal ions can mediate redox reactions by reversible changes in the metal ion’s oxidation state
* ex: carbonic anhydrase
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caarbonic anhydrasse zinc center helps:
metal ion catalysis
* polarize the bound water promoting ionization (with nearby histidine)
* temporarily stabilize the negative charge on the hydroxyl ion
* temporarily stabilizes the negative charge on the newly created HCO 3-
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Michaelis-Menten Kinetics describes...
* special conditions for studying enzyme reactions
* enzyme activity
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MM Kinetics
* Second step of the reaction is catalyzed
* Not reversible
* Rate limiting step -highest activation energy step
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MM
v0
has linear dependence on V max and [S] at really small substrate concentrations
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Km
defined as the [S] where the velocity is equal to one-half the maximal velocity
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Low Km
tight binding to substrate (ES staying together more than breaking apart)
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high Km
weak binding (ES breaking apart more than staying together
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kcat
* describe rate limiting step of any reaction
* equal to the rate constant for the rate limiting step if there is one)—under saturating conditions
* aka turnover number: maximum number of substrate molecules converted to product in a given unit of time
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Competitive inhibition
* Similar in structure to substrate
* Capable of reversible binding to enzyme active site
* Unable to undergo chemical transformation to product
* S and I can never be bound at same time
* In the presence of a competitive inhibitor, it takes a higher substrate concentration to achieve the same velocities that were reached in its absence.
* So while Vmax can still be reached if sufficient substrate is available, one-half Vmax requires a higher [S] than before
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Competitive inhibition
linneweaver-burk
* addition of inhibition causes:
* no change in the y-intercept (1/Vmax) since V max remains unchanged
* increase in the slope ( Km/Vmax) since Km increases
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Uncompetitive inhibition
* inhibitor:
* Binds to some region on enzyme other than active site
* Binds only to ES complex
* Influences activity of enzyme only when [S] and [ES] are high
* A reduction in the effective concentration of the E ∙S complex increases the enzyme's apparent affinity for the substrate leading to a decrease in Km
* It takes longer for the substrate or product to leave the active site resulting in a decreased (Vmax)
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Uncompetitive inhibition-Lineweaver burke
* In a Lineweaver-Burk plot, addition of inhibition causes:
* the y-intercept (1/Vmax) increases since V max value decreases with inhibitor
* the x-intercept (-1/Km) increases since Km value decreases with inhibitor
* Slope (Km/ Vmax) unchanged
* Uncompetitive inhibition works best when substrate concentration is high.
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Mixed or Noncompetitive inhibitor…
1. Binds to some region on enzyme other than active site
2. Presence of inhibitor does not affect substrate binding
3. Does interfere with catalytic functioning of the enzyme
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Noncompetitive inhibition
MM plot
With noncompetitive inhibition, enzyme molecules that have been bound by the inhibitor are taken out of the game so enzyme rate (velocity) is reduced for all values of [S], including Vmax and one-half Vmax.
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Noncompetitive inhibition
LB plot
* In a Lineweaver-Burk plot, addition of inhibition causes:
* The y-intercept (1/Vmax) increases since Vmax value decreases
* The x-intercept (-1/Km) same since Km value unchanged
* Slope (Km/ Vmax)changed for both
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Enzyme regulation types
covalent modification, proteolytic cleavage, allosteric control
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Covalent modification
228
proteolytic cleavage
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allosteric control
230
Best buffer has pKa...
+ 1 of desired pH
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pka on titration curve
* pKa of an acid or base can also be determined experimentally as the midpoint of titration curve
* susceptibility of proton to removal by reaction with a base is described by its pKa value
232
mm
V0 has...
has linear dependence on V max and [S] at really small substrate concentrations
233
steps to solve
234
calculating pI steps
235
myoglobin o2 binding curve
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hemoglobin binding curve
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reading MM plot
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reading LB plot
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mixed inhibition
lb plot
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other about glycine
* small side chain → doesn’t contribute to hydrophobic effect
* only aromatic molecule
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other about methionine
* Slightly nonpolar thioether group
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Isoelectric pH (pI)
pH at which the net electric charge is zero
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secondary structure + ex
stable arrangements of amino acid residues giving rise to recurring structural patterns
α helix, β conformations
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tertiary structure
all aspects of 3D folding of a polypeptide
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Quaternary structures + ex
a protein with 2+ polypeptide subunits
2 major groups: fibrous and globular
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Myoglobin vs hemoglobin fractional saturation curves
* Myoglobin - as [O2] increases, myoglobin becomes saturated very quickly and then levels off
* Myoglobin has a high affinity for oxygen, binds oxygen strongly, and does not release oxygen very easily
* Hemoglobin - hemoglobin has a lower affinity for oxygen, binds oxygen relatively weakly and releases it more easily than myoglobin
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Activation energy
* difference between the energy levels of the ground state and the transition state
* a higher activation energy corresponds to a slower reaction
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Chymotrypsin
* Enhances the rate of peptide bond hydrolysis
* Transient covalent acyl-enzyme intermediate is formed
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List the types of noncovalent interactions that are important in providing stability to the three-dimensional structures of macromolecules. (b) Why is it important that these interactions be noncovalent, rather than covalent, bonds?
Noncovalent interactions include hydrogen bonds, ionic interactions between charged groups, van der Waals interactions, and hydrophobic interactions. (b) Because noncovalent interactions are weak, they can form, break, and re-form more rapidly and with less energy input than can covalent bonds. This is important to maintain the flexibility needed in macromolecules.
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Instant cold packs get cold when the contents, usually solid urea and liquid water, are mixed, producing an aqueous solution of urea. Although this process is clearly spontaneous, the products are colder than the reactants. Explain how this is possible in terms of the difference between Δ*G,* Δ*S* and Δ*H*.
Since we are told that the dissolution reaction is spontaneous, the Δ*G* must be negative. The cold pack reactants are absorbing heat into the system making the product and the surrounding universe cold. This means the since the reaction absorbs heat the Δ*H* must be positive. Given Δ*G* = Δ*H* – TΔ*S*, this is possible if the Δ*S* is very large and positive as well, as one would expect for a solid dissolving, to maintain 2nd thermodynamics law. The 2nd law of thermodynamics is that total entropy of system and surrounding always increases. So entropy increase has to be greater than Δ*H*
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T/F
## Footnote
a) The α-carboxy group is half dissociated at pH 9.67.
b) Glutamate would be a good buffer at pH values near 2.2 and 9.7
c) Glutamate’s pI is between 2.19 and 4.25
a) FALSE THAT IS AROUND PH 2.19
b) YES BECAUSE PKA REGIONS ARE GOOD BUFFERING REGIONS
c) YES BECAUSE ABOVE 4.25 THE -1 CHARGE STARTS TO PREDOMINATE SO HAVE LESS 0 CHARGED SPECIES. ABOVE 2.19 0 CHARGED STARTS TO PREDOMINATE. ACTUAL PI VALUE IS 3.22
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at pH above 6 deprotonation of the carboxylate side chain of poly Glu leads to repulsion between adjacent negatively charged groups which destabilizes the alpha helix and results in unfolding. At pH below 7 protonation of amino group side chains of poly Lys causes repulsion between positively charged groups leading to unfolding. deprotonation of lys R group causes it to become neutral
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alpha keratin has alpha helices that forms coiled coiled structures which have a slightly tighter turn at 5.2 compared to 5.4. Steaming aka heat and stretching will break h bonds holding alpha helices allowing it to be stretched or elongated to resemble a beta strand which is 7 angstroms. Allowing wool to shrink allows for protein to resume its normal alpha helical conformation
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Raw wool is typically combed and stretched in order to be spun into sweaters or socks. When these products are washed in hot water or heated in a dryer, they shrink. Silk, on the other hand, does not shrink under the same conditions. Explain.
silk is already in an extended conformation naturally (beta sheets) so it is resistant to stretching and shrinking. wool can assume an extended conformation when stretched. Moist heat and shrinking allows wool to adapt its native alpha helical structure.
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At low concentrations, the hemoglobin tetramer reversibly dissociates into two α1β1 dimers. Considering the nature of cooperativity in hemoglobin, what would you expect to be the Hill constant for O2 binding to these dimers? Explain
The Hill coefficient would likely be \>1 (*nH* \> 1 indicated positive cooperativity) because the two subunits of the α1β1 dimers can still interact cooperatively. But it is unlikely that *nH* \> 2 since *nH* is typically less that *n* for (the number of O2 binding sites) in this allosteric protein.
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c
## Footnote
B binds tighter because half of its binding sites are occupied at a lower concentration than A. When Y=0.5 that is the Kd
257
A monoclonal antibody binds to G-actin (monomeric) but not to F-actin(polymeric). What does this tell you about the epitope recognized by the antibody?
The epitope is likely to be a structure that is buried when G-actin polymerizes to form F-actin so that’s why it cannot recognize F actin.
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Describe the molecular basis of rigor mortis, which is caused by the lack of ATP store, based on what we learned about muscle movement.
Contraction of muscle is based on the controlled interaction of myosin with actin arranged in filaments. Individual myosin head groups interact with G-actin monomers (of F-actin polymers). In death, ATP stores are quickly depleted preventing the release of the myosin head group from the actin monomer/polymer that usually occurs with ATP binding. With many myosin head groups bound to many actin monomers and no ATP to release the binding interaction the muscles become locked in the position at which the ATP ran out.
259
What amino acid function group of which residue is most likely responsible for the effect of pH?
the histidine residue since it is a major player that donates and receives proton groups which can affect pH.
260
Why would the activity of chymotrypsin increase as the pH increases based on the chymotrypsin mechanism of action.
An increase in pH would render ionizable histidine unprotonated thus allowing to be unprotonated which is what the chymotrypsin mechanism needs for it to act as a general base and accept protons form serine.
261
Cells can develop a resistance to drugs by increasing the cellular concentration of the enzyme that that drug inhibits. If a cell increases its concentration of a given enzyme to 10 times the normal amount, which parameters listed below will be increased ten-fold?
## Footnote
* K*m
* K*I
[S]
* v*0 when [S] = *K*m** **
* k*cat
Catalytic efficiency
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An enzyme has a single active site which it can bind and hydrolyze either X or Y. However, the enzyme cannot bind X and Y at the same time.
1. Will the Km for X be affected if Y is present in the reaction mixture? Explain
2. Will Vmax for X be affected if Y is present in the reaction mixture? Explain
a) Yes. Y is a competitive inhibitor for X; apparent Km for X will increase
b) No the Vmax will be unaffected
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The Pka of an acid depends on pH but also its environment (other amino acids) when apart of protein structure. Predict the effect (pka raised or lowered) of each of the following environmental changes on the pKa of a glutamic acid side chain
A lysine side chain is present.
the pka will be lowered. at certain pH lysine is basic and positively charged so it can stabilize the negative charge of a glutamic acid. The apparent pka will be lowered since a small pka value means easily deprotonated. In this case the pH of the solution didn’t have to take the proton because lysine took it.
264
The Pka of an acid depends on pH but also its environment (other amino acids) when apart of protein structure. Predict the effect (pka raised or lowered) of each of the following environmental changes on the pKa of a glutamic acid side chain
terminal carboxyl group of the protein is brought near it
the pka will raised. the electrostatic repulsion that can occur will disfavor removal of a proton from the carboxyl group and the glutamic acid since they will both be negatively charged. so it disfavors removal and thus apparent pka higher meaning harder to remove proton of glutamic acid.
265
Many isolated enzymes (not in complex with its substrate) will be denatured at 37 degC and inactive. But when its substrate is present the enzyme is active at 37 degC. Suggest a reason as to why this could be.
Stabilization of the enzyme is provided when substrate is bound. Thus raising how well it can adapt to increased temperature like 37 which would normally inactivate it.
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When a polypeptide is in its native conformation, there are weak interactions between its R groups. However, when it is denatured there are similar interactions between the protein groups and water. What then accounts for the greater stability of the native conformation?
In the unfolded polypeptide, there are ordered solvation shells of water around the protein groups. The number of water molecules involved in such ordered shells is reduced when the protein folds, resulting in higher entropy. Hence, the lower free energy of the native conformation. We also have the formation of non covalent interactions lending to the greater stability.
267
Indicate the probable effects of mutating a Cys amino acid to a Met amino acid. Consider R-group size, hydrophobicity, polarity, charge, and/or propensity to take on various secondary structures. Explain your reasoning
* Cysteine is polar and uncharged while methionine is nonpolar.
* Both have a sulfur group but the S of methionine cannot be ionized like cysteine.
* Replacement of Cys with Met adds nonpolarity to that space and takes away the ionizability of that space. so capability to have electrostatic interactions gone.
* If this mutation was on the C or N terminus may actually stabilize if it was an alpha helix since having charged species at the terminals can have destabilizing affects if there were other like charged species.
* Cys can also form disulfide bridges while met cannot so this ability is lost with the mutation which may interrupt necessary disulfide interactions.
* Met (ΔΔG = 0.88) favors alpha helix more than Cys (ΔΔG = 3) based on propensity values (smaller delta delta G value means alpha helix favored) so may be possible to disrupt if secondary structure was alpha helix
