Biochemical Fundamentals Flashcards
(465 cards)
1
Q
Describe the RNA tetraloop-receptor interaction.
A
It begins with a tetraloop (of a hairpin or stem loop) of a conserved GAAA sequence. It forms long-range interactions (most common) with an 11-nt loop motif. The sequences involved in the interaction are conserved, but function can vary based on the RNA structure in which the motifs are found.
2
Q
What is the functional significance of protein acetylation?
A
Resistance to degradation Alters DNA binding properties
3
Q
What drug inhibits xanthine dehydrogenase and why?
A
Allopurinol; Xanthine dehydrogenase converts substrates to uric acid which precipitate in joints causing gout. Allopurinol prevents the formation of uric acid.
4
Q
What is the protein modification site for acetylation?
A
The N-terminus.
5
Q
What does carboxylation modification to glutamate residues achieve functionally?
A
Binding of Ca2+ ions
6
Q
How can enzyme kinetics be measured?
A
By measuring the rate of the reaction with changing substrate concentrations.
7
Q
Describe O-linked glycoproteins that remain in the intracellular space.
A
They consist of a single monosaccharide addition to a threonine, tyrosine, or serine hydroxy residue and is reversible.
8
Q
WHy do we draw the Hawarth Projection rather than a carbohydrate as a chair conformation?
A
For simplicity.
9
Q
What type of glycosidic bond links phosphate species?
A
O-glycosidic
10
Q
Is Co-IP performed in vivo or in vitro?
A
In vitro, typically from cell lysates where all proteins are present.
11
Q
What residues of the ribonuclease enzyme (hydrolysis of RNA) interact with uracil of an RNA substrate?
A
Thr-45 and Ser-123. This makes sense because these side chains readily form hydrogen bonds and can be ionized.
12
Q
Which proteins are N-glycosylated and where does it occur?
A
Only those destined for secreation or for the membrane. N-glycosylation occurs in the endoplasmic reticulum.
13
Q
How do intergrin interactions inside and outside the cell modulate one another?
A
Talin protein binds to actin on the inside of the cell, causing conformational changes in the Beta-subunit of the integrin on the outside of the cell and changes in integrin interaction with EC matrix conmponents.
14
Q
What is an example of triple-helix fibrous proteins?
A
Extreme tensile strength.
15
Q
How can SPR be used to measure which residues of IL5Ralpha are necessary for binding of IL5 monomer?
A
Mutagenesis can be performed on certain residues followed by SPR to see whether the 1:1 ratio iof binding is conserved.
16
Q
What are modification sites on proteins for lipid attachment?
A
Myristoyl group to N-terminus Glycosyl-phosphatidylinositol and Farnesyl to C-terminus Palmitoyl group to Cysteine or Serine
17
Q
How does the myoglobin knockout affect land animals?
A
There is no phenotypic difference, meaning that hemoglobin has evolved to function similarly/more effectively.
18
Q
Describe CO (carbon monoxide) poisoning.
A
CO binds heme 200x more strongly than O2 and thus competes for the binding position. Therefore, tissue hypoxia ensues along with headache and dizziness.
19
Q
What characteristic of a protein largely determines its structure?
A
Amino acid sequence.
20
Q
Define Km in terms of Vmax.
A
It is the substrate concentration at which half of Vmax is reached.
21
Q
What are the functions of GAGs and proteoglycans?
A
They exist in the ECM to protect the cell and provide cushion, and form a protective coat around microbes.
22
Q
Show by expression that velocity is proportional to substrate concentration when substrate concentration is low (in an enzymatically-catalyzed reaction).
A
23
Q
What is an alternative to CD4 mimicry to inhibit HIV-1 and T cell membrane fusion?
A
We can mimic what happens in the energy cascade by “springing the moustrap” before HIV-1 fuses to T cell on its own. We can do this by diffusing the envelope and stopping infection from occurring. HNG, a triazole gp120 inhibitor, restructures the envelope protein gp120 so that it inhibits binding to both CD4 and 17b coreceptor surrogate.
24
Q
Describe base pairing in RNA stems or helices.
A
They tend to be complementary but are not strictly so.
25
What is velocity in terms of Vmax, substrate concentration, and Km?
26
What does the nomenclature of specifying a disaccharide include?
Must start at the reducing sugar, include the sugar type, the position of the anomeric carbon (beta or alpha), the location of the glycosidic bond, the type of epimer (D or L).
27
Describe SCID and the role played by enzyme defficiency.
Severe Combined Immunodeficiency Disease: adenosine deaminase is defective, so there is no ability to amount an immune response. It is sometimes treated by gene replacement therapy.
28
How many RNA strands are involved in a 3-way junction?
Three RNA strands with linking sequences between the junctions.
29
Describe how ITC can measure the stoichiometry of IL5 and IL5Ralpha?
ITC measures the change in heat characteristic of binding activities (binding gives off heat, exergonic) when ligand is injected into the sample cell which contains the protein receptor. The molar ratio at the break point shows that just one IL5 monomer (wild type composed of dimer) is necessary for binding to IL5Ralpha.
30
What allows for the high degree of DNA bending around histones to form the nucleosome?
The T:A base step has the lowest stacking energy and can therefore be broken to induce more deformation of the DNA.
31
Which feature of a peptide besides residues must be known to determine structure?
The angles of the phi and psi bonds.
32
Do allosteric enzymes exhibit hyperbolic or sigmoidal kinetics?
Sigmoidal.
33
Define ligand.
Any small molecule bound to a macromolecule.
34
What is one of the commonly discussed least specific enzymes?
Protease.
35
What functions are N-glycosylation responsible for (hint: 5).
Protein folding, stability of folded protein, recognition events (e.g. cell-cell), immune system evasion (e.g. capsid of HIV), targeting of proteins to different cellular compartments (e.g. proteosome).
36
How many different stereoisomers are there for a carbohydrate?
2^n where n is the number of assymetrical carbons.
37
What determines the rate of formation of glycosylated Hb?
Glucose levels.
38
What is the double reciprocal of the Michaelis-Menton equation?
Lineweaver-burke.
39
Describe Co-IP.
It is a bait-prey method in which an antibody is immobilized on a solid phase bead (hook) and equilibrated with the antigen being studied, the bait (can be anything, such as a protein). Then cell lysate is run over the beads (liquid phase) which may or may not contain "prey", or molecules which interact with the prey. Then, the beads are precipitated and studied to see what prey bound to the bait.
40
How does N-linked glycosylation occur?
The nitrogen is derived from the side chain of asparagine.
41
Why do ligands and DNA-binding proteins preferentially bind the major groove of B DNA?
The major groove is wide and deep while the minor groove is narrow and deep. Thus, the major groove has more volume for binding (exception - TATA-binding protein/TBP)
42
When substrate concentration is very high, what does the Vmax of an enzyme-catalyzed reaction depend on?
Enzyme concentration
43
Describe the function of GroEL chaperone.
An aggregation prone peptide binds to the central channel of GroEL together with the co-chaperonin GroES. Subsequent ATP hydrolysis triggers the release of both the polypeptide and GroES back into solution, when the polypeptide obtains its native conformation or rebinds to the chaperonin if it is still aggregation-prone.
44
Which covalent protein modification helps protect secreted proteins?
Glycosylation.
45
What do phosphorylation modifications to proteins help achieve?
It functions to regulate the protein.
46
How would a polar mutaiton of one of the residues in the binding pocket of hemoglobin affect blood color?
It will brown the blood, as water will be able to access and oxidize Fe, preventing the binding of O2 (characteristic of methemoglobinemia).
47
The removal of what group causes acyl formation?
The removal of a hydroxyl group from an oxoacid.
48
Which are the basic amino acids?
Histidine, lysine, arginine.
49
What is the rotation between beta and alpha anomer called?
Mutarotation.
50
How do IDP adapt to different binding partners?
They can fold into different 3D conformations.
51
What type of protein can read the carbohydrate code?
Lectins.
52
Which angle of a peptide is the ф bond?
The alpha carbon-nitrogen bond.
53
Describe specific base catalysis.
Involves quick and complete removal of proton from substrate.
54
What properties of a reaction determine the direction in which it progresses at equilibrium?
The starting and ending free energies of the reaction (this is why enzymes cannot change the direction the a rxn will progress).
55
What causes the microheterogeneity of glycosylation?
The fact that the same protein or lipid can be modified by different carbohydrates. This is where the study of glycomics, or the sugar code, arises.
56
What does it mean that hemoglobin is a dimer of dimers?
It is a tetramer composed of a2B2 (mostly helices, each with its own prothetic group).
57
What forces stabilize the deoxy form of Hb, and how can these be broken?
The deoxy (T) state is stabilized by salt bridges between alpha and beta subunits of Hb. Because they are ionic interactions, they can be broken by pH changes, causing change to R state. Therefore, inducing salt bridges in the R state can also cause the release of O2 and transition to T state.
58
What is pyranose?
59
Describe the "substrate reaction type" enzyme nomenclature.
The substrate name followed by the type of reaction followed by "ase". Does not tell us the co-substrate. e.g. alcohol dehydrogenase (does not indicate the cosubstrate, NAD+)
60
How is the presence of uracil in DNA fixed if it occurs?
Uracil DNA glycosylases recognize the problem and excise uracil if they find it in DNA.
61
What is the Vmax in terms of enzyme concentration for a reaction with two steps?
62
How does poryphoryn ring of heme group function to bind O2?
The four nitrogens of the ring of heme prosthetic group coordinate an iron atom with up to 6 coordination bonds (four from the ring and one from O2).
63
How can entropy always be increasing if the binding of a flexible peptide to a regid enzyme is entropically unfavorable?
The binding may disrupt the ordering of water spheres, for example, so the overall entropy of the system increases.
64
Where are L- and D-amino acid oxidases found and which one is active?
Only L-amino acids will be oxidized, and they are present in the brain and kidney.
65
What does sulfation and carboxylation modification to proteins achieve?
It helps keep proteins in solution.
66
What is the organ of glucose homeostasis?
The liver. As glucose concentrations decrease, it traps less glucose in the liver cells, causing glucose to be shunted to the brain where there is a higher affinity for its entrapment in brain cells.
67
Why isn't uracil used in DNA?
Spontaneous deamination of cytosine results in a nucleobase identical to uracil, potentially resulting in a mutation of the complementary guanine to adenine in the next round of DNA replication if uracil were used in DNA.
68
Activation energy is a property of the ______ of a rxn, and thus enzymes find a better ______ to lower the activation energy (same word).
Mechanism.
69
Describe the reaction performed by a dehydrogenase.
Redox reaction. Electrons are transferred in the form of hydride from one substrate to another, where the accepting substrate is not oxygen.
70
Describe the reaction performed by an oxidase.
A redox reaction in which electrons are transferred to O2 in the form of hydride.
71
What are proteins that need chaperone assistance for folding prone to?
Aggregation.
72
What activities might induce a sickle cell anemia attack and why?
Activities that promote the deoxy state of HbS, such as increased altitude, increase in BPG, exercise. The oxy state is not a problem, because valine is not exposed in this conformation.
73
Describe the ligands that bind DNA covalently.
These binders are irreversible and include cis-platin.
74
Which are the aromatic amino acids?
Phenylalanine, tyrosine, tryptophan.
75
Describe the amino acid and phenotypic change involved in sickle cell anemia.
A valine is substituted for a glutamate in the beta subunit position 6 of Hb, resulting in an ionic to hydrophobic change in polarity. Therefore, when valine is exposed in the deoxy state of Hb, the subunits polymerize to reduce valine exposure to water. The polymerization causes branching and unflexible fibers.
76
What is a common example of a protein isoform?
Creatine kinase, found in both the muscle and used to diagnose myocardial infarction, exists in 3 different isoforms.
77
what is a polyhydroxy molecule?
a sting of carbons with hydroxyl groups
78
How is proline an unusual amino acid?
It has no rotation around the peptide bond. The N-term is part of a ring and can therefore not participate in hydrogen bonding. Usually causes a kink in the protein. Found in turns.
79
What characteristic amino acid residues are the heme O2 binding pocket normally surrounded by?
It is normally surrounded by hydrophobic amino acids so that water is excluded and will not interfere with the oxidation state of Fe and thus O2 binding.
80
Where is dideoxyribose used?
In Sanger sequencing.
81
How do peptide triazoles hijack env gp120 of HIV-1 plasticity and virus metastability to kill the virus?
82
What is a coiled coil?
It is an a-helix that participates in protein-protein interactions.
83
Recombinant protein engineering shows similar tolerance of sequence variation. What does this say about protein scaffolding?
It shows that as long as the conformational scaffold can still form then complementary binding surfaces will not be disrupted (e.g. conservation of a helix).
84
What effect does carbohydrate modification have when it is O-linked versus N-linked?
They have starkly different functions.
85
What are holoenzymes?
Holoenzymes are conjugated enzymes.
86
What is a molten globule protein intermediate?
It is an intermediate formed formed from the cooperatively folded protein domains. They go on to equilibrate to the protein's native conformation. Polypeptide chains with secondary structures formed but with a looser tertiary structure than the native state is said to be in Molten Globule State.
87
Which stacking pair has the lowest stacking energy?
(TA)(TA), the lowest TA step
88
What is pyranose?
A six-carbon sugar.
89
How does the isoelectric point relate to pH?
The isoelectric point, pI, is the pH at which the net charge of a species is zero. Thus, when pH \< pI, a peptide has a positive charge. When pH \> pI, a peptide has a negative charge.
90
How does the intramolecular cyclization of monosaccharides create two new stereoisomers?
The carbonyl carbon, hemiacetal or hemiketal, becomes either beta or alpha (carbonyl hydroxy either pointing up or down).
91
Describe subunits of fetal Hb.
HbF is composed of a2y2 (alpha and gamma)
92
Describe the hypothesis for the contrasting effects of cPT irreversible binding inhibitors, CD4mc inhibitors/sensitizers, and BMS/18A-class allosteric inhibitors on the modular conformational components of HIV-1 gp120.
When gp120 trimer is open, its beta loop sticks out at binding site #2, placing gp120 in a closed conformation. Here, BMS/18A reversible inhibitors binds, stabilizing the closed conformation of gp120. cPT irreversible inhibitors bind both site #2 and site#1, the latter of which activates the inward opening of the beta loop, causing the bridging sheet and allowing for coreceptor binding to gp120. However, because site #2 is bound as well, gp120 only *thinks* it is in the open conformation, resulting in its shedding from gp41. The CD4mc inhibitors/sensitizers bind only site #1, causing bridging sheet and activation of the coreceptor.
93
How is histidine used as a biological switch?
It has a pKa very close to neutral pH, so with changes in pH it can have different charges, causing it to interact differently with other species.
94
Define the reaction catalyzed by hydroxylase.
It is a 3-substrate reaction involving O2 in which a protonated substrate is oxidized by the first O atom to form a hydroxyl group, and the second atom is reduced to H2O, often by NADH.
95
Describe the SPR mutagenesis analysis of IL5 for IL5Ralpha interaction.
Bound to the biosensor surface is an anti V5 antibody, to which the IL5Ralpha (IL5 receptor) is bound (solid phase). The analyte containing IL5 is then passed over the chip. When the charged residues (glutamic acid, aspartic acid) in the 4-helix bundle of IL5 are mutated, there is a loss in binding to the receptor. However, not all of the positive charged residues are responsible for receptor alpha binding. The mutation of Glu 13 disrupts binding to receptor beta-c
96
Why are HIV-1 envelope-based vaccines and generation of neutralizing immunogenic epitopes challenging?
They are challenged by conformational plasticity:
- Conformational masking: gp120 is very flexible, so the epitopes are conformationally masked that could trigger the immune system to make antibodies.
- glycosylation masking: epitopes of gp120 are masking by glycosylation, preventing detection by the immune system.
- Tolerance to mutational variation: as long as the scaffolding is unaffected, HIV-1 can tolerate changes in its coding sequence.
97
What is Ka (Kw) and pH of pure water?
98
What are the common IgG antibody scaffolds?
H2L2
99
How is Km and Vmax affected by noncompeititve inhibitors?
Vmax is decreases because catalytic activity is inhibited, but Km does not change because the active site affinity for the substrate is no different.
100
What is the modification site of hydroxylation to proteins?
-Carbon of certain Proline residues
101
Where do glycosidic bonds occur?
At the hydroxyl group of the anomeric carbon by replacement with nitrogen (N-bonds) or at other positions (O-bonds).
102
Describe the protein folding pathway.
It is initiated upon immediately upon translation by reversible and rapid formation of local secondary structures. Secondary structures then form domains through the cooperative aggregation of folding nuclei. Domains finally form the final protein through “Molten globule” intermediates. Molten globules equilibrate to their final native structure.
103
Where do glycolipids tend to be found in the cell?
As parts of the cell membrane.
104
What is the structure of collagen?
It is a polyproline type II helix where number of repeating residues is 3. Gly-X-Pro. Often contains hydroxyproline.
105
define enantiomer
Same formula, but mirror images. Can only be two enantiomers in a pair. D or L.
106
To which ring, the five-member or six-member, of purines does the N-glycosidic bond form?
It binds to N9 of the five-member ring.
107
What do most drugs on the market target?
Enzymes. This is due to prevent nonspecific interactions with the drug and affect other metabolic pathways.
108
Which are the amide amino acids?
Asparagine, glutamine.
109
Describe an important and common example of an RNA protein complex as a macromolecular machine?
Ribosome.
110
Why is myoglobin and hemoglobin evolutionarily required?
O2 is not readily soluble, so aerobic organisms need a transport system. Myoglobin, a single chain protein, serves to facilitate the diffusion of oxygen in muscle (especially to the mitochondria). Hemoglobin, a tetrameric protein comprised of 4 myoglobin-like chains, is used to shuttle oxygen from the lungs to the tissues and return protons and carbon dioxide to the lungs.
111
What is the origin of the HNG triazole gp120 inhibitor?
It began as a low potency polypeptide. The internal proline was was converted to a triazole using click chemistry and a copper catalyst, increasing the potency of the peptide. Then, the triazole was cyclized into a metabolically stable, drug-like molecule.
112
What type of interactions are found in the active site?
Among catalytic groups there are electrostatic interactions, hydrogen bonding, and hydrophobic interactions to exclude water.
113
What is the functional significance of proteolytic processing?
It functions in the activation of proenzymes.
114
What type of glycosidic bond links nucleotide species?
N-glycosidic bond.
115
What is the role of the 2'-OH group in RNA?
It allows a spot for extra hydrogen bonding (potential for two new H-bonds per nucleotide = increased stability)
116
what functional group does ketose have? aldose?
ketonie; aldehyde
117
What enzyme state (T or R) is favored by equilibrium when an allosteric activator binds the R state?
The R state.
118
What oxidation state is iron in Ferrihemoglobin/Ferrimyoglobin and how does it affect oxygen binding?
It is the 3+ oxidation state. Oxygen cannot bind because water coordinates with Fe here.
119
Which purine is adenine and which is guanine?
1. is adenine; 2. is guanine
120
Describe site-directed mutagenesis by recombinant redesign.
Insert normal gene into plasmid cloning vector, changing the nucleotide sequence of a potentially important amino acid. Add a synthetic primer containing the mutated sequence. DNA polymerase and ligase finish replication. Plasmid is then introduced to cells (e.g. E. coli) where replication and division of cells occurs. Phenotypic effect is then monitored (i.e. by SPR).
121
What is the prothetic group in hemoglobin used to bind O2?
It is heme, which includes an iron atom.
122
How might we go after latent T cell reservoirs infected with HIV-1?
Figure out how to get the envelope proteins of HIV-1 to express themselves on the T cell surface, which can then be targeted by the immune system.
123
Describe the minor groove (concave) of the three forms of DNA that cause available space for binding to differ.
A DNA minor groove is wide and shallow, B DNA is narrow and deep, Z DNA is narrow and deep.
124
Describe the kissing loop RNA tertiary structure.
The loops of two stem-loops or hairpins have complementary bases that form long-range base pairings, stabilizing the structure of both loops.
125
What are the basic forces driving protein interaction?
Complementary surfaces driven by pre-formed or induced conformational scaffolds; stabilization of protein complexes uses forces similar to those in protein folding (Hydrogen bonds, van der waals interactions)
126
What is a protein isoform?
Any of several different forms of the same protein. They can arise from slightly different genes or alternative splicing of the same gene.
127
Which type of base catalysis is used by enzymes and why?
General base catalysis. Partial proton removal. Amino acids are weak acids and bases. Occurs at physiological pH. Not as fast as specific base catalysis, but fast enough for the reaction to occur.
128
What type of protein is a triple-helix, and what are some examples?
A fibrous protein. Some examples include collagen, tropomyosin, keratin.
129
Which are the nucleophilic amino acids?
Serine, threonine, cysteine
130
Describe the Lineweaver-Burk plot in competitive enzyme inhibition.
The slope increases with inhibitor concentration, while y-intercept (Vmax) remains the same. -1/Km becomes less negative.
131
Describe from binding to fusion, the interaction of HIV-1 to T cells.
gp120 first binds with CD4 of the T cell, causing a gp120 conformational change and rearrangement allowing for higher affinity to the T cell coreceptor to gp120. An even greater gp120 conformational change occurs, causing the envelope protein to slide up and reveal the gp41 6-helix bundle near the T cell membrane. The helix bundle binds to both the T cell via the fusion protein and remains on the HIV-1 envelope, fusing the two membranes together like a mousetrap. The energy initially stored in the envelope membrane is used to catalyze the fusion.
132
What protein type is responsible for a virus recognizing its host?
Lectins present on the virus capsid will recognize glycoproteins on the host's cell surface.
133
Why might hyperglycemic mother might not deliver as much O2?
Glycosylation interferes with BPG binding and thus Hb will remain in the R state longer, binding O2 more tightly. This may be dangerous for a mother carrying a fetus.
134
What is the core structure of the porphyrin ring in heme?
A porphyrin is a heterocyclic macrocycle made from 4 pyrrole subunits linked on opposite sides (α position) through 4 methene bridges (=CH-)
135
Describe the cooperative binding of O2 to Hb after the first O2 binds to the deoxy (T) conformation.
Globin units of deoxyHb are tightly held by electrostatic bonds in a T state. O2 binding imposes chemical and mechanical stresses that ‘break’ these bonds, leading to the R state.
136
How many basepairs make up human DNA?
Approximately 3 billion base pairs.
137
What does the middle of a titration curve represent, for an acid for example?
This is the midpoint, at which half of the acid is dissociated. This is where concentration of products and reactions are equal, so pH equals the pka.
138
Explain how the hydrophobic effect is spontaneous
The formation of water spheres around hydrophobic molecules is entropically unfavorable, so the coming together of hydrophobic species disrupts this. This is a more entropically favorable reaction.
139
Describe pathway feedback regulation via protein interaction in the context of aspartate transcarbamylase (ATCase).
ATCase forms cytidine triphosphate (CTP) from aspartate and carbamyl phosphate. Measurement of the ATCase reaction rate with varying aspartate concentrations shows that when CTP is added, a sigmoidal curve appears, showing that CTP binds ATCase cooperatively to inhibit it. Addition of ATP shows a curve shift to the left, indicating that it cooperatively activates the ATCase. This is possible due to multiple subunit binding sites on ATCase.
140
What role do vitamins play in catalysis?
They are precursors for coenzymes.
141
How does rapid metabolism signal for the transition of oxy to deoxy state of Hb?
Rapid metabolism produces high amounts of CO2 and H+, the latter of which changes pH. This causes for salt bridges to be formed and transition to T state of Hb so that CO2 can be carried to the lungs for release.
142
Describe formation of the first tetrahedral intermediate of chymotrypsin.
The first tetrahedral intermediate forms immediately after the formation of the enzyme-substrate complex of chympotrypsin. Here, there are four bonds to what was the carbonyl carbon of the substrate residue. Once oxygen of serine becomes involved in the tetrahedral intermediate, it no longer has an affinity for the hydrogen that was part of its hydroxyl group, so the hydrogen is entirely bound by the histidine imidazole nitrogen. Next, the electrons now on the intermediate's carbonyl oxygen are pushed back in for the formation of a new amino group on the peptide. Since a negatively charged amino group would be a bad leaving group, the protonated imidazole acts as a weak acid in general acid catalysis to provide the proton to make the amino group a good leaving group.
143
Describe the bonds of the transition state for the enzyme-substrate complex of chymotrypsin.
Partial single bond where substrate carbonyl double bond to oxygen was, partial bond between imidazole nitrogen of His57 and hydrogen of Ser195, and partial bond between Ser195 oxide and substrate carbonyl carbon (with all of the respective bonds partially broken).
144
What does lipid attachment to proteins assist with?
Insertion into the membrane.
145
Describe the allosteric inhibition of HIV-1 gp120 by peptide triazoles (such as HNG) in terms of the conformation of gp120.
The peptide triazole binds to gp120, trapping it in a conformational state which does not allow it to move down its free energy cascade.
146
Who received the Nobel Prize for the discovery of catalytic RNAs in 1989?
Altman and Cech.
147
How do DNA-binding proteins read sequences in the DNA major groove?
They read sequences through hydrogen bonds and hydrophobic contacts. Every base pair in the major groove has a unique pattern. In the minor groove, however, A +T and G + C have the same patterns.
148
How is BPG produced by erythrocytes?
Erythrocytes synthesize and degrade the 2,3-BPG by a diversion of the glycolytic pathway.
149
What are endopeptidases and what is a common example?
Proteases that cleave a protein within the amino acid sequence. One example is chympotrypsin.
150
What are reducing sugars and how do they function?
Only aldehydes are redox reactive, because they can be either oxidized (turns to carboxylic acid) or reduced (turned to alcohol). They are not active when they are cyclized, so not all of these species in solution will be active.
151
Difference between nucleotide and nucleoside, and example of nomenclature.
Nucleotiside is just the ribose sugar and nucleobase (e.g. adenosine/deoxyadenosine) and nucleotide is a nucleoside + phosphate group(s) (e.g. adenosine triphosphate/deoxyadenosine triphosphate)
152
What type of curve/kinetics is followed by non-allosterically regulated enzymes?
Hyperbolic or Michaelis-Menton kinetics.
153
Describe the release of the C-terminal peptide substrate from the serine of chymotrypsin second tetrahedral intermediate.
Electrons from what was the acyl oxygen in the previous step are pushed back into the tetrahedral carbon, kicking out the serine side chain. The histidine imidazole acts as a weak acid in general acid catalysis, providing the proton to which the serine side chain oxygen binds to to make it a better leaving group (as opposed to the nucleophile in the formation of the enzyme-substrate complex).
154
What is free energy at equilibrium?
G=0
155
What does insufficient hydroxylation of collagen lead to?
Scurvy.
156
How might a reaction overcome a competitive inhibitor?
Max out the concentration of susbtrate to outcompete the inhibitor.
157
Describe the Fab of IgG and the hypervariability regions.
The Fab, or the homologous scaffolding region, consists of 4 beta sheets, the end of which is the hypervariable loop where binding specificity is determined based on sequence.
158
Which are the small amino acids?
Glycine, alanine
159
Describe the resulting kinterics data from SPR experiments.
The first half of the curve shows association of prey with bait over time, while the second half shows dissociation of prey from bait when a buffer is passed over the chip. The k*off* divided by the k*on* tells us the Kd of the interaction.
160
What pathways cause the production of acidic byproducts from actively metabolizing muscle tissue?
glycolysis and fatty acid oxidation
161
Which asparagine residues are glycosylated?
It depends on the sequence context. Typically, Asn residues in the sequence Asn-X-Ser or Asn-X-Thr can be glycosylated. The asparagine must also be accessible by glycosyl transferase (i.e. an Asn reside at the center of a protein may not be glycosylated).
162
What is rate acceleration of an enzyme?
It is the reaction rate + the enzyme divided by the reaction rate without the enzyme. The larger the rate, the more effective the enzyme.
163
Describe the class of heterotropic allosteric interactions.
Nonequivialant ligands. Binding of substrate at active site and binding of allosteric inhibitor/activator at allosteric site.
164
What is the precursor to the coenzyme pyroxidal phosphate?
Pyroxidal also known as vitamin B6. This coeznyme is involved in amino acid transformation reactions.
165
Describe specific acid catalysis.
It is a form of catalysis in which the pH is decreases where strong acids quickly and completely protonate a substrate.
166
Describe the role of group II introns and to what molecule they are the ancestor
They are large ribozymes that catalyze their own splicing, and are the ancestor to the eukaryotic yeast and human spliceosome. First, the intron cleaves the 5' end, then the 3' end, then ligates them together. These events are all catalyzed at the same active site.
167
Describe the role of base triples in RNA folding, and describe the pairing of the triple GCC.
They allow for structures such as tetraloop-receptors to form. Mostly used in the formation of RNA tertiary structures. GCC would involve complementary binding of G to C, and the binding of the second C to the Hoogsteen edge of the base paired C.
168
Which form of DNA is found under high salt conditions?
Z DNA
169
What is the typical Km range for enzyme-catalyzed reactions?
10^-1 to 10^-6 M (the lower the Km, the stronger the association).
170
Describe the hydrolysis of the C-terminal peptide substrate from the acyl-enzyme chymotrypsin.
The second substrate, H2O, is deprotonated by a general base catalysis again by histidine imidazole nitrogen (new positive charge again stabilizaed by Asp) so that it can act as a strong nucleophile by which to attack the acyl carbonyl. In transition state water will form partial bond to the carbonyl, and C-O double bond will be partially converted to a single bond, forming the second tetrahedral intermediate.
171
What are the two broad classes of DNA-binding proteins and given examples?
Sequence-specific interactions (e.g. regulatory proteins/txn factors) and nonspecific interactions (e.g. nucleases and DNase1)
172
Other than having a larger Km than hexokinase to control liver-brain glucose homeostasis, how else is glucokinase regulated?
It is regulated by proteolytic degradation during starvation as well as de novo synthesis during the fed state.
173
What is the first enzyme to be regulated in a given metabolic pathway?
The enzyme which catalyzes the first irreversible reaction specific to that pathway.
174
define diastereomer
same formula, not a mirror image. not an enantiomer
175
Why does BPG production increase in erythrocytes at high altitudes?
The ability for the body to deliver O2 to tissues naturally decreases with altitude, so red blood cells compensate by increasing BPG production and thus O2 release from Hb.
176
What curve shape indicates cooperative folding of a protein?
A sigmoidal curve.
177
Where in the cell are Il5Ralpha and IL5Rbeta-c found?
They are integral membrane proteins, so study of their interactions can lead to better insight into biological processes and intervention (drug) outcome.
178
Why is it feasible to engineer certain leucine zipper proteins to control expression of specific genes?
Because leucine zippers bind *specific* nucleotide sequences in the major groove.
179
What kinetic value does the velocity equal when substrate concentration is very large (enzymatic reaction)?
Vmax
180
What secondary structure comprises myoglobic?
Mainly helices (80%)
181
Describe the formation of the enzyme-substrate complex for chymotrypsin.
It occurs by general base catalysis and nucleophilic covalent catalysis. Serine first nucleophilically attacks the carbonyl of the substrate. This occurs quickly because His57 imidazole deprotonates hydroxyl of Ser195, making it a very strong nucleophile and allowing the attack to proceed quickly. The removal of the proton proceeds by general base catalysis, as the imidazole nitrogen of His57 is a weak base. The Asp residue (negatively charged) facilitates the new positive charge on the His imidazole.
182
What are the chemical similarities among all nucleobases?
They are planar and all have a nitrogen acting as a hydrogen bond donor or acceptor.
183
How do homologous proteins from different species show preservation of function despite sequence variation?
As long as scaffolds can form and binding sites are preserved, the homologous proteins will exhibit the same function (e.g. hemoglobin)
184
What does the sigmoidal curve of O2 binding to Hb show about its binding behavior?
It shows that O2 binding is cooperative. Also shows that at pO2 of 50, Hb can drop off O2 at capillaries whereas Mb cannot.
185
Why does HbF require gamma subunits, and how do they differ from beta subunits?
The fetus requires a different hemoglobin, Hb F, because it has to absorb oxygen from the placenta where the oxygen tension is lower than in the lungs. The acetylation of some N-terminal histidines prevents binding of BPG, allowing for tighter O2 binding and less release.
186
Because the HIV-1 envelope is a moving target built to be reorganized and activated upon receptor interaction, how might the envelope be targeted by a broadly active inhibitor?
gp120 could be targeted by a mimic of CD4 containing the Phe of the binding site that would be able to access the gp120.
187
At which spot do deoxyribose and ribose differ?
At the 2' carbon.
188
Describe the mechanism of Hb allostery between R and T states.
In the deoxy (T) state of Hb, the distal His E7 pushes Fe out of the plane with the heme porphyryn ring so that it is not adequately coordinated for O2 binding. In the oxy (R) state, the same residue pulls Fe back into the plane with heme so it can bind O2. This causes the conformational change of Hb.
189
What factors precipitate sickle cell crisis?
All things that induce deoxy conformation -- Hypoxia • Fever • Vascular stasis • Respiratory viruses • Acidosis • Dehydration (reduction of blood volume)
190
Why is the pO2 of the arterial end of capillaries so much higher than that of the venous end?
So that Hb can drop of O2 at cells and not have it picked back up again.
191
Describe binding where the allosteric activator is the substrate.
Binding of one subunit active site in the T (tense) state induces the relaxed (R) state, allowing for more of the substrate to bind the other active sites. This is cooperative binding.
192
Describe the proteolytic processing that results in functional chymotrypsin.
Chymotrypsin begins as chymotrypsinogen (zymogen) which is cleaved by trypsin, leading to active chymotrypsin.
193
What are helix bundles?
Supersecondary protein structures that consist of 3 or more helices packed together.
194
What is the helical sense for the 3 forms of DNA?
A DNA is right handed, B DNA is right handed, and Z DNA is left handed.
195
What type of effector is BPG?
It is an allosteric effector.
196
Besides protein cleavage, what two reaction types can chymotrypsin catalyze?
Ester hydrolysis and amide hydrolysis.
197
On which step does the rate of the enzyme-catalyzed reaction depend?
It depends on the second step, as it is not in equiliibrium. In a large reaction scheme, this is the step that would be highly regulated.
198
Why is amino acid conformation important?
For amino acids that become parts of proteins and enzymes, active sites are stereospecific.
199
Which components of this chymotrypsin reaction scheme can be isolated and why?
All but the transition states, because they have bonds partially formed and partially broken, and these are in very high energy states. The instability of the TS is essential for the rxn to progress downhill.
200
Why do enzyme conformational changes occur and why are they important?
In some cases, it may be to exclude other things in solution such as water. But, on the whole, enzyme catalyzed reactions occur in many steps, each requiring different binding interactions and thus conformational changes.
201
What are lipopolysaccharides and what response do they ilicit?
They ilicit an immune response (toxic shock syndrome) because they are a bacterial glycolipidconjugate. (similar to LPS in humans, but has a different oligosaccharide)
202
What was the first family of chaperones discovered?
Heat shock proteins.
203
Describe "substrate only" enzyme nomenclature.
The ending, "ase" is added to the name of the substrate. e.g. urease.
204
What are the envelope proteins of HIV-1 responsible for membrane fusion?
gp120, a docking glycoprotein, and gp41,a transmembrane glycoprotein. These are the only virus-specific components on the envelope membrane.
205
What portion of the RNA sequence in secondary structure determines how much movement the structure is capable of?
The length of the loop (in the case of loops or hairpins) or of the linker sequence (in the case of junctions).
206
Which stacking pair has the highest stacking energy?
(GC)(GC)
207
Which enzyme state, R or T, is favored at equilibrium when an allosteric inhibitor binds the T state?
The T state.
208
How can protein binding sites be mapped?
A combination of mutagenesis (does sequence changes also change function?) and and binding analysis.
209
Which form of gp120 of HIV-1 is in the ground state? The liganded or unliganded form?
Unliganded by CD4.
210
Describe chymotrypsin-catalyzed hydrolysis reactions.
They are broadly specific. Involve nucleophilic addition across carbonyl carbon.
211
What is a common example of a coenzyme organic prosthetic group?
FAD is involved in oxidation reduction reactions and is an intermediate carrier of electrons (i.e. can be reduced and then oxidized to return to initial state). Can act with dehydrogenase which removes hydride (negative charged hydrogen)
212
Describe the conformation of the N-glycosidic linkage in nucleotides.
The bond is stereospecific, meaning the base is always attached to the same side of the plane as the C5 carbon of the ribose. Also, rotation of the N-glycosidic bond gives rise to anti and syn conformations, with nucleobase groups either pointing away (most common due to decreased steric hinderance) or towards the 5' carbon.
213
Where do chaperones bind proteins?
They bind the exposed hydrophobic regions of partially folded proteins. They may also assist protein transport into cellular organelles.
214
What does the rightward shift of the saturation curve during the Bohr effect with decresed pH tell us about O2 release?
It shows that O2 will be released at higher pO2 because the T states is stabilized. It also shows that it takes higher pO2 to saturate Hb.
215
At what point is a buffer most affective?
At a pH + or - 1 from its pka.
216
Which amino acids can be O-glycosylated?
The same ones that can be phosphorylated, Ser, Tyr, Thr.
217
How do helices of helix bundles interact by knobs into holes packing?
Side chains of nonpolar residues mesh together. This is allowed due to the slight distortion of the individual helices and the inclination of their axes with respect to each other.
218
Which enzyme is the exception to stereospecificity?
Racemases.
219
What does turnover number represent?
The number of substrate molecules turned into product by one enzyme molecules in one second.
220
How was the structure of Hemoglobin determined?
Because it is plentiful and easy to purify, it was able to be X-ray crystallized. The X-ray hits each and every atom and diffracts. The diffraction pattern allows for the structure to be deduced.
221
Describe DNA packaging in eukaryotes.
It is packaged in the nucleus with the help of histone proteins. Histones are postively charged, so they bind DNA very tightly. When DNA is wrapped around an octameric complex of eight histone proteins (H2A, H2B, H3, H4) a nucleosome is formed.
222
Why is GC content important when designing primers?
Because GC bonding is stronger than AT, you want to make sure the primer is stable enough in solution but not too stable.
223
What is the mechanism of the Bohr effect?
Salt bridges are stabilized by carbamate formation to N-term of Hb subunits.
224
What are the two types of O-linked glycoproteins?
Those that are secreted such as mucins synthesized in the golgi secretory pathway (very large) and those that remain in the cell, such as O-GlcNAc.
225
Describe the tetranucleotide hypothesis.
It shows that nucleotides are connected only by phosphates and that sugars are not involved.
226
What is the Levinthal paradox?
It is a thought experiment which considered whether protein folding is random. It was determined that for a protein of only 100 amino acids with just 3 rotational positions, that it would take 3^100 combinations to get to the correct one, if they were tried randomly.
227
Desribe the chymotrypsin enzyme-product complex.
The enzyme returns to ithe state it was in before forming the enzyme-substrate complex. Serine has a hydroxyl group and the positively charged histidine imidazole nitrogen is stabilized both by the hydroxyl hydrogen and Asp negative charge. The second product contains a carbonyl just as the substrate did.
228
Describe the lock and key hypothesis for an enzyme and its substrate.
States that the active site and the substrate are rigid and explains the specificity of one for the other. However, negative experimental evidence was found that when rigid, no reaction occurs.
229
Describe the conformation of pentose sugars in nucleic acids.
They are nonpolar and exist in two predominant ring conformations in nucleic acid structure: 2'-endo and 3'-endo. In 2'-endo the 2' carbon is displaced from the median plane, and in the 3'-endo conformation the 3' carbon is puckered.
230
Why do the R groups of proteins matter in hydrolysis reactions by chymotrypsin?
Because they determine specificity of binding at active site.
231
Why is base stacking in DNA highly favorable?
It minimizes the contact of nonpolar atoms in bases with water.
232
Which family of enzymes require divalent metal cofactors and how do they function?
Kinases require these cofactors. They form a complex with the negatively charged phosphate of ATP, thus activating the phosphate and making it easier to transfer to the substrate. These metal cofactors are not consumed and are thus also not cosubstrates.
233
What are glycoconjugates?
They are carbohydrates linked to other molecules such as proteins/peptides or lipids.
234
What are glycoforms?
They are the same protein with different oligosaccharide modifications.
235
Which bond of a peptide is the Ψ bond?
The alpha carbon-carbonyl carbon bond.
236
what factors are responsible for the behavior of water? (e.g. can form many weak interactions that amount to strong overall interaction)
High electronegativity difference between oxygen and hydrogen, high melting and boilng points
237
Describe the Lineweaver-Burk plot for noncompeititve inhibition.
With increasing inhibitor concentration, the slope becomes more steep. The X-intercept -1/Km does not change, but the y-intercept 1/Vmax increases (correlating to decrease in Vmax).
238
Describe the interaction between HTH binding motif with B DNA.
One alpha helix binds through hydrogen bonds and hydrophobic interactions to exposed bases in the major groove, while a second alpha helix stabilizes the interaction.
239
In which RNA tertiary structure does RNA form a binding site for small molecules?
Riboswitch.
240
What are riboswitches? Explain in the context of the fluoride riboswitch.
They are a tertiary RNA structure which senses the presence or absence of certain metabolites and responds by regulating gene expression in bacteria. In the absence of a specific metabolite, here fluoride, the switching sequence, upstream of the gene expression platform is switched down, or on, and bacteria are free to grow. When the fluoride metabolite is present and binds the 5' UTR region of the riboswitch, the switching sequence is switched up, or off, inhbiting the growth of bacteria by turning off the expression of certain genes.
241
What is an example of a protein complex "machine" involved in transcription?
Transcription initiation complex.
242
What is K at equilibrium of a reaction?
243
How can carbohydrates be acetylated?
Hydroxy groups can be modified by amino groups, which can then be acetylated.
244
What is the functional significance of glycosylation in protein folding?
It signals proteins for binding with chaperones.
245
Describe an experiment that favors the chymotrypsin hydrolysis reaction that proves the formation of the acyl-enzyme intermediate and that it is labile.
Can take advantage of the fact that chymotrypsin only functions at neutral pH. Radiolabel the enzyme and work it at neutral pH. Drop the pH to 4, where some enzymes should be trapped as the acyl-enzyme. Use gel filtration to separate products and intermediates and screen for radioactivity. Will see that the protein is radiolabelled. Can then raise pH back up to see that activity resumes and radioactivity dissipates at a rate expected for enzyme activity, proving that results are not an artifact of lowering the pH.
246
What happens to a deoxy Hb crystal when it is oxygenated, and what does it suggest about Hb structure?
It shatters, suggesting that oxy and deoxy Hb exist in two different conformational states.
247
Which type of glycosylation exists in the cytosol and nucleus?
O-linked glycosylation.
248
Which order reaction does an enzyme-catalyzed reaction that is no longer dependent on substrate concentration represent?
It represents a zero-order reaction. This occurs at the saturation point.
249
What are the two types of reversible enzyme inhibition?
Competitive and noncompetitive (both bound noncovalently)
250
What is activation energy?
The difference in free energy between the ground and transition states of a reaction.
251
Describe the electrostatic potential of the major groove in the three DNA forms.
A DNA major groove is strongly negative, B DNA is negative, and Z DNA is neutral/positive (makes sense due to salt concentrations)
252
What is the precursor to the coenzyme FAD?
Riboflavin, or vitamin B2. This is also a precursor to FMN.
253
Which form of DNA is found in dehydrated conditions, and is the favored structure in RNA molecules?
A DNA
254
What is a common coenzyme cosubstrate involved in redox reactions?
NAD+. The cosubstrate undergoes chemical change (reduction) and does not have to return to its original state, a difference from organic prosthetic groups.
255
Which conformations do oxy and deoxy Hb exist in, and what are their respective affinities for O2 binding?
Oxy Hb is in the R state and binds O2 strongly, whereas deoxy Hb is in the T state and has poor affinity for O2 binding. These conformations exist in equilibrium.
256
Which amino acids have chiral centers?
All of them except for glycine. This is why nature has chosen one conformation, and naturally that is the L conformation.
257
What do sequence-specific DNA-binding proteins look for when binding?
They look at the sequence for patterns of potential hydrogen bonding, and they look at the shape readout which may be distorted by certain sequences of bases. Proteins look here to know where to bind.
258
How do carbohydrates achieve diversity beyond number of carbons and stereoisomers?
Functional group modifications
259
What is the functional significance of proline hydroxylation in proteins?
Maturation and secretion of collagen
260
What is the clinical significance of BPG?
Donated blood stored in acid-citrate-dextrose, gains O2 binding affinity, due to a loss of natural BPG levels • Transfused red cells, totally depleted of BPG, can regain normal levels within 24 hrs…too long • Must artifically reconstitute BPG levels before transfusion.
261
Are protein-protein interactions in a network single proteins interacting or multi-protein complexes interacting?
Most often they are multi-protein complexes interacting.
262
What is a prothetic group?
A non-amino acid substance that is strongly bound to a protein and necessary for the protein portion of an enzyme to function.
263
Which are the hydrophobic amino acids?
Valine, leucine, isoleucine, proline, methionine.
264
Describe the reaction catalyzed by racemase.
It is responsible for forming a racemic mixture of L- and D- isomers.
265
Why do phosphatases not operate as the reverse of a kinase?
Whereas kinases begin with ATP and transfer a phosphate group to their substrate, phosphatases do not form ATP from the phosphate removed from its substrate.
266
What is the equation for free energy at equilibrium?
267
The rate constant of which step determines the turnover (Vmax/[Etotal]) of an enzyme-catalyzed reaction?
k3
268
What is a zinc finger?
It is a supersecondary protein structure exactly large enough to fit in the major groove of DNA. Involved in DNA binding, oftentimes transcription factors. Composed of helices.
269
Name the bond type that connects nucleotides.
Phosphodiester bond between 5' phosphate and 3' hydroxyl group.
270
Describe noncovalent DNA ligands and where they bind.
These are reversible binders, and they bind to the major groove, minor groove, and intercalate between bases.
271
What is the relationship between free energy and K?
272
Which life cycle enzymes of HIV-1 are inhibited in HAART therapy?
Integrase, reverse transcriptase, and protease are inhibited by HAART therapy, but this is not a cure.
273
What does the distance in numbering of the catalytic residues of chymotrypsin tell us about enzyme structure?
It tells us that the rest of the enzyme, the scaffolding, is essential for bringing the residues of the active site together.
274
What is a protective heteropolymer carbohydrate?
GAGs and proteoglycans
275
Which is the most common form of DNA found in aqueous solutions?
B DNA.
276
What portion of an enzyme does the active site typically make up?
Just a very small portion. The rest of the protein acts as a scaffold to bring the active site or stabilizing components closer together.
277
What is IL5 and what is its significance?
It is interleukin-5, and it is a cytokine that matures into an esinophil, causing allergic inflammation and asthma. Figuring out how to disrupt its interactions with its receptors, responsible for maturation to esinophil, is helpful in drug design.
278
How much CO2 is transported with Hb compared to O2?
97% O2 transported with Hb, slightly more than 10% of the CO2 transported with Hb. [80% of the carbon dioxide is transported as bicarbonate]
279
Describe the induced bending of DNA in terms of the common example of the TATA-binding protein (TBP)
TBP binds extensively in the minor groove of B DNA. Lys and Arg residues interact with the phosphate backbone, while bulky and planar side chain sticks into the minor groove, opening it up. It is easy for TATA rich sequences in the minor groove to be opened up and induce DNA bending because of the weak hydrogen bonding between these base pairs. Also the stacking between these base pairs is weaker.
280
What is the storage homopolymer in mammals?
Glycogen.
281
Describe the usefulness of Hb glycation.
Though about 6% of HbA in humans is glycated, a greater percentage can indicate and help diagnose diabetes.
282
What oxidation state is Ferrohemoglobin/Ferromyoglobin, and how does it affect oxygen binding?
It is the 2+ oxidation state, and can readily bind oxygen.
283
How and why does BPG alter O2 binding to Hb?
BPG (2,3-Bisphosphoglycerate) decreases Hb affinity for O2, increasing delivery of O2 to tissues and shifting the saturation curve to the right. Because it is a highly negative molecule, it greatly stabilizes the deoxy state of Hb so that affinity for O2 decreases.
284
What is the experimental purpose of suicide enzymes?
To determine the function/important components of an enzyme by inhibiting its function.
285
Define metabolism.
All reactions in a cell occurring at a given time.
286
How can enzymes act as reagents in clinical measurements?
Kidney function is measured using creatinine levels. Cardiac damage can be measured by presence of creatine phosphokinase.
287
What two conformations does the alpha chiral carbon of amino acids have, and what is the one exception?
L and D amino acids. Glycine does not have a chiral alpha carbon, so it only has one conformation.
288
Describe "trivial" enzyme nomenclature.
In this form of nomenclature, there are no rules. e.g. chymotrypsin.
289
What is the slope of the Lineweaver-Burk plot?
It is Km/Vmax
290
Why do we not call beta and alpha anomers epimers?
Because they readily convert between one another. Epimers cannot interconvert.
291
How can you determine whether an ionizable group of an amino acid is charged at a certain pH?
Consider its pKa.
292
Describe basic leucine zipper (bZIP) as an example of DNA sequence-specific interaction.
Leucine (hydrophobic) does not interact with the DNA, but with each other on opposite sides of the zipper to stabilize the protein. The residues that come in contact with DNA at the major groove are Arg, Lys (positive) through hydrogen bonds, though hydrophobic interactions can take place at other zipper resideues.
293
How can a study of protein primary structure be evolutionarily advantageous?
We can look at sequence homologies across different organisms to predict function. We can use it to identify molecular pathologies, such as sickle cell anemia. It can help us understand potential function-structure relationships.
294
Are metal cofactors such as Mg+ inorganic prosthetic groups, and why?
They are not because they are not covalently attached to the enzyme. They are necessary for the reaction, however
295
How are proteins glycogen phosphorylase and glycogen synthase, phosphorylated by glycogen hormone, affected by phosphorylation?
Glyogen phosphorylase is active in order to break down glycogen for the rest of the body, while glycogen synthase is inhibited by phosphorylation.
296
What modification type does O-glycosylation compete with, and what is the benefit of the competition?
Phosphorylation; it helps the cell to fine tune certain signals.
297
What role does distal His E7 of hemoglobin protein play in O2 binding to heme?
It guides O2 into the binding pocket, allowing O2 to form the final and 6th coordination bond with iron.
298
What do chaperones prevent?
"Dead end" folding pathways. (i.e. those that lead to no function)
299
What type of interactions are formed between allosteric inhibitors/activators and their enzymes?
All noncovalent interactions.
300
Describe the formation of a-helices and the types of residues found in them.
There are about 3.6 amino acid residues per turn of the helic, and all H-bonds occur vertically (as opposed to B-sheets). Hydrogen bonds occur between residues 3.6 amino acids away from one another. Most amino acids other than proline (can't turn), glycine (too flexible), and tryptophan (too large) can be found in a-helices.
301
How does the sigmoidal curve for allosteric interactions change with inhibitors and activators? In other words, how does rate change?
In the presence of activators, velocity sharply increases at low substrate concentrations (looks a bit more hyperbolic). In the presence of inhibitors, there is a large lag in velocity increase with substrate concentration increase. This appears more sigmoidal.
302
What is the Bohr effect?
It is when an acidic environment or adding carbon dioxide to the blood reduces the oxygen-binding affinity of Hb because the T state is stabilized. This causes O2 to get released.
303
Describe the reaction catalyzed by kinase.
In the presence of a divalent metal ion such as Mg2+, kinases catalyze the covalent addition of phosphate to a hydroxyl group.
304
What is a common example of a heat shock protein?
Hsp-70
305
What is the rate law for first order reactions?
306
What drug inhibits dihydrofolate reductase and why?
Methotrexate; purpose of inhibition is to prevent formation of dTTP and thus DNA synthesis and cell division in cancer cells.
307
Which interactions hold together tertiary structure scaffold (used to create complementary contact surfaces from small portions of the amino acid sequence)?
Mostly noncovalent interactions.
308
What does hemoglobin transport?
O2, CO2, and H+.
309
What forces drive the formation of large, machine like protein complexes?
Modifications (often ATP or GTP) and conformational changes.
310
What does it mean for active site binding to be multoplicative?
That all of the binding interactions together are much stronger than the sum of the individual interactions.
311
Where is hemoglobin found in the body?
Primarily in the red blood cells (erythrocytes).
312
What bond type gives rise to polysacchatides from monosaccharides?
glycosidic bonds
313
How many zinc fingers does protein SP1 have?
3.
314
Express velocity when substrate concentration is equal to Km.
v = Vmax/2
315
Describe the two types of prosthetic groups found on conjugated enzymes.
Neither type is part of the peptide chain. There are organic prothetic groups such as heme in hemoglobin, and there are inorganic groups such as FeS clusters.
316
Describe the formation of the chymotrypsin acyl-enzyme.
The reaction has passed the transition state from the tetrhedral intermediate to the acyl-enzyme. The free amino terminus from the substrate has left as the first product after being protonated (partially at first) by histidine imidazole nitrogen.
317
Describe the energy difference between the unfolded, transition, and folded states of a protein.
While the unfolded and folded states differ only slightly in their free energies, the transition state is much higher, requiring the assistance of chaperones in some cases.
318
Describe intercalators and a well-known example found in DNA gel staining.
they are planar heteroaromatic rings that stack between adjacent nucleotide bases (also planar). They are stabilized by pi-pi interactions and van der waals forces, much like nucleobase stacking. Ethidium bromide is a classic example of an intercalator.
319
Describe RNA primary, seconday, and tertiary structure.
Primary structure is the linear sequence. Secondary structure is the pairing of bases (not always complementary). Tertiary structure is folding into a unique 3D shape without the help of protein binding.
320
How can interactions and thus conformational changes of proteins be quantified in the cell using FRET?
FRET = fluoresence resonance energy transfer. Similar to yeast two-hybridization, a target protein is recombinatorally bound to a blue fluoresent protein, and the ligand is bound to a green fluoresent protein. When bound, the excitation of the blue fluorophore will in turn excite the green fluorphore. Violet light excited blue fluorophore (lblue emitted) and blue light excites the green fluorophore (green emitted). So, if violet light is shone on the cell and blue light is emitted, then just the blue fluorophore is excited and the proteins are not bound. However, if violet light is shone on the cell and green light is emitted, then the proteins have been bound. This light emitted can be measured to quantify the amount of binding.
321
Vmax and Km are constant when which player in an enzyme-catalyzed reaction is constant?
Enzyme concentration.
322
What is the first step of a reaction catalyzed by an enzyme?
The formaion of a noncovalent, reversible enzyme-substrate complex.
323
Which gas ion mediates salt bridges in the Bohr effect?
Cl-
324
Describe the experiment that led to the discovery of spontaneous protein refolding.
Ribonuclease A was subject to B-mercaptoethanol to reduce all 4 of its disulfide bonds which are in place to stabilize the protein. Urea was also used to denature the protein to a random coil. Removal of the denaturants showed that the ribonuclease became once again catalytically active, meaning it must have refolded to the proper conformation. This proved that the help of an enzyme is not always needed, and that the "code" for protein structure is within the amino acid sequence.
325
Describe intrinsically disordered proteins (IDP) and which residues they tend to be composed of.
They contain protein segments which lack definable structure, giving them much flexibility. Thus, they are composed of amino acids lys, arg, glu, and pro which confer more flexibility.
326
Describe binding of O2 to myoglobin.
Because there is only one unit, there can be no cooperativity of O2 binding. It is either bound to heme or it is not.
327
Define the "systematic" enzyme nomenclature.
The name of all substrates are listed, followed by one of six main reaction types to whcih "ase" is added as a suffix. Each enzyme is also assigned a classification number. e.g. alcohol:NAD+ oxidoreductase EC1.1.1.1
328
Compare adult and fetal saturation curves for O2 binding to Hb.
The fetal curve is leftward of the mother's. This shows that a higher saturation point of Hb must be reached in the fetus for O2 to be released from Hb. In other words, a lower pO2 is required to saturate fetal Hb.
329
Describe surface plasmon resonance (SPR) as a biosensor for detecting binding (studying protein interactions) of a site-directed amino acid mutant.
It is another type of bait-prey binding experiment, basing success of binding on changes in mass. Dextran hydrogel containing bait molecule (solid phase) is attached to a gold chip. and the analyte (prey) is flooded across in the liquid phase. Polarized light is shown through a prism at the base of the chip, and the refractive index is measured, indicating changes in binding. Binding causes an increase in the refractive index on the chip surface. This causes the the resonance angle for plasmon induction to become altered, which can be measured with a detector.
330
Where among IgG antibodies does sequence changes change binding specificity, and in which structures is there homology?
Antigen binding sites are within Fv domains of Fab fragments. Fab fragment structure, circled, is homologous among all antibodies (thus the scaffold does not change). However, sequence changes at hypervariable regions will change the antigen which the IgG binds.
331
Describe the temporal regulation of Hb genes throughout development.
There is full beta subunit expression 18 weeks post-birth.
332
How is the peptide bond formed?
Nucleophilic attack of N-terminal nitrogen on C-terminal carbonyl carbon.
333
What distinguishes enzymes from other catalysts?
Their chemical structure is primarily protein and RNA (ribozymes), they have enormous rate accelerations, and they have a very high degree of specificity (unlike metal catalysts, for example with bind nonspecifically).
334
Describe the activation of chymotrypsinogen by proteolytic cleavage.
Chymotrypsinogen is only active during digestion. Trypsin hydrolyzes between Arg and Ile of chymotrypsinogen, inducing a conformational change (though the two halves of the newly formed pi-chymotrypsin are still connected by sulfide bridge). Chymotrypsin then hydrolyzes and removes from pi-chymotrypsin a serine-arginine residue and a threonine aspartame residue, leaving behind 3 covalently attacked (via cysteine) polypeptides. These disulfide-linked polypeptide chains make up the active alpha-chymotrypsin.
335
Describe the transition of RNA secondary structure to stable tertiary structure and the role of metal ions.
Ions condense (sometimes proteins) onto the RNA secondary structure, neutralizing more than 90% of the charge. The molecule collapses to form a compact intermediate. The molecule then searches for the most stable conformation.
336
Describe the interactions and structures that allow for protein binding to A form RNA.
Non canonical base pairing, base pair triples, and adjacent loops distort the A form RNA by causing the minor groove to open up, thereby facilitating specific protein interactions.
337
What composes the "picket fence" around heme?
Nonpolar amino acids. Acts as a fence to keep out water and the oxidation of ferrous to ferric state of Fe.
338
Name the following pyrimidines.
1. cytosine; 2. thymine; 3. uracil
339
How can protein binding specificity vary within homologous scaffolding sequences?
As long as scaffolding overall is uninterupted, sequence variation at specific loci can introduce ability to change binding ability (e.g. IgG antibodies)
340
What is the fischer projection and what does it accomplish?
carbohydrate structure representation to avoid having to define stereochemistry. The horizontal arms come out of the plane, and the vertical arms go into the plane.
341
For an enzyme-catalyzed reaction, the tangental slope at low substrate concentration of a velocity vs. substrate graph appears to be what order uncatalyzed reaction?
It appears to be first-order.
342
What are the two ways in which coenzymes can function?
Organic prosthetic groups (not all of these are considered coenzymes) and cosubstrates (but not all cosubstrates are considered coenzymes either).
343
What is the difference between the two coenzymes types, organic prosthetic groups and cosubstrates?
The prosthetic groups are part of the enzyme and thus must remain from the beginning to the end of the reaction. The cosubstrate can leave after the reaction has completed.
344
Describe the induced fit hypothesis for an enzyme and its substrate.
When the substrate binds, it induces a catalytically active change in conformation of the active site. X-ray crystallography data support this hypothesis.
345
In which conformation are amide bonds most stable?
Trans conformation (due to steric conflict of alpha carbons in the cis conformation. Can happen with glycine or alanine, however).
346
How do enzymes change to equilibrium constant, K?
Enzymes cannot change K, they just allow the reaction to progress to equilibrium faster.
347
Describe the formation of pseudoknot RNA tertiary structures.
They are made form long-range base pairing interactions. They only *look* like knots, and form in the absence of divalent metal ions.
348
Describe the major groove (concave) of the three forms of DNA that cause available space for binding to differ.
A DNA major groove is narrow and deep, B DNA is wide and deep, and Z DNA is flattened.
349
How many alpha-helices does tropomyosin have?
Four.
350
Why is the peptide bond so short?
Because it has double bond properties (resonance). (it is sp2)
351
Which forces are involved in base stacking?
van der waals and pi-electron cloud interactions (hydriphobic bonding). (bases are parallel)
352
What is the CO2 stabilizing effect of Hb?
CO2 in the blood can bind covalently to the N-terminus to form a carbamate (negatively charged) and H+, helping to stabilize salt bridges in T state (deoxy) (Presence of H+ is a signal that O2 needs to be picked up in the lungs).
353
What is the rate law for pseudo first order reactions?
354
How is feedback inhibition an example of allosteric inhibition?
Because the product at the end of a pathway, G for example, is much different than the starting substrate, A, it cannot bind to the active site and must therefore be a heterotropic allosteric activator.
355
Which is the first regulated enzyme in glycolysis and how?
Phosphofructokinase in the phosphorylation of fructose-6-phosphate. AMP acts as a heterotropic allosteric activator, as it indicates a lack of ATP in the cell.
356
What drug inhibits protease and why?
Protease inhibitor; purpose of inhibition is to prevent replication of HIV virus in AIDS patients.
357
Describe the class of homotropic allosteric interactions.
One substrate affects the binding of another, identical substrate.
358
Why is it difficult to reveal the structural nature of the env trimer target (gp120) of HIV-1?
Stabilized trimers used for structure determination may not be the same as the native mature unliganded state.
359
How does the henderson hasselbach equation relate pH to pKa?
360
Explain the cyclization of monosaccharides.
The hydroxyl group initiates a nucleophilic attack on the carbonyl carbon (sp2 --\> sp3, a reversible reduction). The resulting carbon species is called a hemiacetal or hemiketal.
361
What are lectins?
There are many different types of lectins and they are proteins that recognize and bind glycoproteins.
362
Describe how peptide triazoles and peptide triazole thiols can cause not only inhibition of infection by binding gp120, but also HIV-1 gp120 shedding and virolysis?
Both types of triazoles cause conformational changes in gp120 which actually cause it to fall off the envelope membrane surface, inactivating the virus. Peptide triazole thiol specifically causes p24 release from the virus as well, which makes up the capsid cone. Therefore, because p24 is natively on the inside of this means PT thiol causes the boring of holes in the membrane surface of HIV-1, allowing p24 to leak out.
363
What are two examples of supersecondary protein structure?
Coiled coil, zinc finger.
364
What is an example of irreversible enzyme inhibition?
Suicide substrate (covalent binding)
365
How do RNA molecules exclude water and form a more hydrophobic environment for folding?
Because H2O can bond with the bases, RNA needs a more hydrophobic environment for folding. Therefore, they utilize base stacking within the helix and in loops.
366
Define zymogen/proenzyme.
It is the inactive form of an enzyme, requiring peptide cleavage for activation.
367
What is an apoenzyme?
It is an enzyme that lacks the prosthetic group that allows it to be catalytically active.
368
How are proteins glycogen phosphorylase and glycogen synthase, dephosphorylated by insulin hormone, affected by phosphorylation?
In the dephosphorylated form, glycogen synthase is active so that sugar in blood can be stored as glycogen. Glycogen phosphorylase is inhibited by dephosphorylation because it does not need to break down glycogen.
369
What is the role of metal ions in RNA structure and function?
- They influence conformational changes during folding and stabilize conformational states by neutralizing the negative charge of the RNA backbone, allowing RNA to fold without help of proteins.
- They organize active sites.
- they activate catalytic residues
- they directly participate in catalysis
370
Describe the a-chain Bohr effect.
The terminal amino groups of the a-chains interact with the carboxy terminal groups of the other a-chain. This proximity increases the pK a of the terminal amino group, thereby, permitting it to form the salt bridge mediated by a chloride ion
371
What are the two classes of exopeptidases and what do they do?
Carboxypeptidase (cleaves from the C-terminus of a protein) and aminopeptidases (cleaves from the N-terminus of a protein). These proteases cleave one amino acid residue at a time.
372
How can aldoses be used to detect diabetes?
Sugars with aldehydes can be reduced to form alcohols, which react with Benedict's reagent (a copper reagent) which detects glucose.
373
What protein composes the capsid core of HIV-1?
gp24 proteins
374
What is furanose?
A five-member ring
375
Are hemiacetals redox reactive? Hemiketals?
Hemiacetals are not redox reactive, but they will be eventually if the solution is in equilibrium (only the linear form is redox reactive). Hemiketals are not redox reactive, nor are the linear ketones.
376
What does the sigmoidal shape of allosteric enzymes tell about the reaction at small substrate concentrations?
It shows that large substrate concentrations are required to reach high velocity.
377
Describe the reaction catalyzed by isomerase.
The isomerase catalyzes the conversion of one isomer to another. e.g. ketone to aldehyde sugar.
378
What structures are myoglobin found in?
Cardiac and skeletal muscles.
379
How do catalysts affect free energy?
They do not effect free energy, but they lower the activation energy, or the energy required to reach the transition state of a reaction.
380
Describe structure determination of the small ubiquitin-related modifier (SUMO).
It has ordered regions (middle) and disordered regions (ends). These disordered regions can become ordered when they bind to one of their complementary partners.
381
Describe the sugar pucker conformation for the three forms of DNA?
A form is 3'-endo, B DNA is 2'-endo, and Z form is 2'-endo at cytosine and 3'-endo at guanine. These differences in conformation allow the backbone to have different shapes.
382
Which types of catalysis are utilized by chymotrypsin?
General acid, general base, and covalent catalysis.
383
What is the function of covalent catalysis between enzyme and substrate?
It activates the substrate for interaction with the second substrate.
384
How can isomerases aid in protein folding?
One example is the cis-trans isomerase converting trans peptide bonds that preceed a proline to cis peptide bonds.
385
In what molecules are 6-way RNA junctions found?
Ribosomes.
386
Define epimer.
Sugar that differs at one position (glucose versus mannose). Ths is also technically a diastereomer
387
Describe the formation of the ribose zipper RNA tertiary structure.
It forms from interactions between the backbones of two RNA strands, specifically by hydrogen bonding between the 2' hydroxyl groups of ribose of antiparallel strands.
388
For an uncatalyzed first order kinetics (single substrate reaction), what does the slope of a velocity versus substrate concentration graph represent?
It represents the rate constant, k.
389
Why is ethidium bromide fluoresent only when it binds to DNA (reversible)?
EtBr is only weakly fluoresent in aqueous solutions, because water quenches the fluoresence. However, when it intercalates beween adjacent base pairs, the hydrophobic environment shields EtBr from water and stabilizes its excited state, allowing it to fluoresce 30-fold higher.
390
Which type of acid catalysis do enzymes use and why?
General acid catalysis, because it involves the partial protonation of weak acids at physiological pH. This is not as fast as specific acid catalysis, but is still significant becaue amino acids are weak acids.
391
What are the two requirements for reducing sugars to be redox reactive?
They must be linear aldoses, and must be in equilobrium with cyclized carbohydrate (e.g. a methylated hemiacetal can no longer be in equilibrium).
392
How is carbon monoxide poisoning (often from cigarette smoking) treated?
It is treated with high pO2 to resaturate heme with O2 for delivery to tissues.
393
Which Hb resides account for the Bohr effect?
The carboxy-terminal His residues in the B - chains (His 146) and the amino groups of the a- chains are responsible for H + transport and thus the Bohr effect.
394
Describe DAPI as a minor groove DNA-binding ligand.
DAPI binds DNA reversibly to stain nuclei. It consists of linked aromatic rings which gives it partial planar character while maintaining flexibility by the linking bond. They are crescent-shaped so they can reach the minor groove, and targets A-T rich regions because they allow the minor groove to be opened up more (disrption of H-bonding and base stacking).
395
Express Vmax in terms of enzyme concentration when substrate concentration is very large.
396
Identify and describe an in vivo bait-prey method.
Interaction partner identification using yeast two-hybrid screening. Here, bait consists of a DNA-binding domain recombinantly attached to a target protein of interest. Also recombinantly expressed in the yeast cell is the prey, a transcriptional activator protein attached to the target protein's putative binding partner. If and when the target and bnding partners come together, so will the DNA-binding domain and transcriptional activator protein, allowing for the activation of a reporter gene and the expression of a reporter protein which can be measured in the cell.
397
For an enzyme-catalyzed reaction with multiple substrates where enzyme concentration is constant, the reaction will depend on which substrate?
The limiting substrate. If a\>\>\>b, the reaction will depend on b and we will get a Kb.
398
How might prolines exist in fibrous structures, and what is an example?
They may participate in helices, just not alpha helices. One example is synthetic collagen-like peptide, pro-gly-gly.
399
What is the role in enzymes in breakdown of urea?
Urease has extreme specificity (catalyzes no other reaction) and catalyzes the hydrolysis of urea by water to form CO2.
400
Which are the three broad types of RNA enzymes or ribozymes?
Group I introns, group II introns, and hammerhead ribozyme.
401
What is the physiological role of chymotrypsin?
Protein hydrolysis in digestion.
402
What is the rate law for second order reactions?
403
How can players in metabolic activity be activated or deactivated?
By modifications, like addition or breakage of high energy bonds (e.g. phsphoryl bonds, addition of nucleotides such as UDP)
404
Why are small quantities of enzymes sufficient to allow for physiological reactions to proceed?
Because enzymes are not consumed in reactions.
405
What is the difficulty of trying to inhibit HIV-1 through small molecule CD4 mimics?
CD4 mimics, such as the organic compound EGLV, do decrease binding of gp120 to CD4. However, the binding of the mimic to gp120 still activates it, allowing it to bind the coreceptor. In these experiments, a coreceptor surrogate Fab 17b is used to measure binding by SPR. This makes the CD4 mimic an agonist rather than an inhibitor.
406
Explain the glycoconjugate portions of glycophosphatidyl inositol (GPI)
GPI anchors proteins to lipids in the cell membrane using a carbohydrate liner between the two. This is only possible when the linker is a reducing sugar.
407
How do forward and reverse rate constants relate at equilibrium?
408
How might the RNA secondary structure of internal loop arise?
It might arise if two non-pairing bases are across from one another. However, RNA does not have to strictly pair with their complements.
409
How does the equilibrium constant relate to the rate constants for the forward and reverse rxns, and what does it tell us about enzyme activity?
Because equilibrium constant cannot change, the enzyme must increase the rate of both the forward and reverse reactions.
410
Which state controls the rate of the reaction overall and is involved in enzyme kinetics?
Transition state.
411
What role do lectins play in inflammation response?
Lectins contribute to the immune response by flagging down white blood cells to the site. Lectins recognize the glycoprotein specific to leukocytes in the blood vessels and bind, slowing down the leukocytes as they approach the site of inflammation.
412
An increase in Cl- in red blood cells will stabilize which conformation of Hb?
The T-state, because it stabilizes salt bridges between the alpha-chains of Hb.
413
What type of kinetics (hyperbolic vs sigmoidal) are followed by noncompetitive enzyme inhibition?
Hyperbolic, or Michaelis-Menton
414
How are enzyme conformational changes modeled by thermophilic enzymes?
At room temperature, enzymes are too rigid and do not catalyze reactions. As temperature is increased, they relax and can function.
415
What are the major forces stabilizing DNA structure?
Hydrogen bonding and base stacking.
416
Describe direct vs diffuse RNA-metal binding.
in direct binding, the ion binds directly to the oxygen atoms of the phosphate backbone. In diffuse bonding, the metal ion bonds to water molecules which bond to the oxygen atoms of the phosphate backbone.
417
How is the Michaelis-Menton curve affected in compeititve inhibition?
The Km increases (requires more substrate to reach same Vmax), but Vmax does not change (Vmax is a result of enzyme concentration and the mechanism of catalysis). The Michaelis-Menton hyperbolic curve shifts right.
418
How can the Km of a reaction be determined without experimentally testing saturating concentrations of substrate?
The inverse of velocity with substrate can be plotted (Lineweaver-Burke) to determine Vmax and Km by the y- and x-intercepts respectively.
419
420
Describe sickle cell trait.
It is the inheritance of a single recessive sickle cell allele. It causes expression of 1:1 HbA:HbS hemoglobin and provides malaria resistance.
421
Which enzyme has a turnover number of 10^7?
Catalase. This is extremely fast.
422
Why can the transition state never be isolated?
It only exists on the order of fs because it is so high energy and unstable.
423
Why is phosphorylation similar to allosteric regulation but not exactly the same?
Because it is a covalent modification, while allosteric interactions are noncovalent. It is similar in that the modification causes activation or deactivation of the enzyme due to conformational change.
424
Which are the acidic amino acids?
Aspartic acid, glutamic acid (protonated below their pka)
425
Why do starting and ending free energies of a rxn not change even in the presence of an enzyme?
These are basic thermodynamic properties of a reaction.
426
How are secondary protein structures formed?
Hydrogen bonding between the backbone components (no R groups involved).
427
Which components of HIV-1 entry into the host cell parallel to components of a mouse trap system?
CD4 of the cell is the mouse, gp120 of HIV-1 is the cheese, and gp41 of HIV-1 is the spring.
428
Describe the coaxial stacking of RNA helices at junctions and how it is thermodynamically favorable.
~0.5-3 kcal/mol free energy is released with coaxial stacking. Helices with shorter linkers tend to be stacked because the linker is less flexible.
429
How many subunits and active sites are allosteric enzymes composed of?
More than one subunit, each with its own active/catalytic site.
430
Which fibrous proteins have alpha-helices?
Tropomyosin and a-keratin. They must be amphipathic in order to pack together.
431
To which nitrogen of pyrimidines does the N-glycosidic bond form?
To N1.
432
What do antibodies require for correct folding?
The assistance of a chaperone.
433
How do polysaccharides glycogen and cellulose differ?
The position of the glycosidic bond.
434
To which residue in the CD4 binding pocket does gp120 bind?
Phe43.
435
How does titrating an acid with strong base affect equilibrium?
It will pull equilibrium towards products, as the base pulls protons out of solution.
436
What type of glycosidic bond connects two sugars?
O-glycosidic bond
437
Molecular definition of carbohydrate
(CH2O)n
438
What are the two adult hemoglobins and their subunits?
HbA (a2B2) and HbA2 (a2d2)
439
Which points on the saturation curve show O2 and H+ release with pH?
440
Does covalent catalysis or uncatalyzed reaction require more steps?
Covalent catalysis
441
What are the two different schemes by which noncompeititve inhibitors inhibit formation of product from substrate?
They can bind the enzyme, preventing substrate binding by changing the conformation of teh active site, or they can bind the enzyme-sustrate complex, preventing the catalytic mechanism.
442
Why does RNA adopt the A form helix?
The extra hydroxyl group at 2' carbon causes steric clashing.
443
Define isozymes.
Enzymes that catalyze the same reaction, but are chemically different or expressed from different genes.
444
Describe chromosomal organization of Hb subunits.
Subunits a and B are on two different chromosomes (16 and 11).
445
What is unusual about cysteine?
The sulfur group is an oxidant and can use its oxidizing ability to form covalent bonds. This earns it the term "molecular staple."
446
In a FRET experiment, if both green and blue light is measured, what would that mean?
That there might be some bleed through of blue light because not all of the target protein is bound by the ligand.
447
Describe B-turns.
Essentially a kink in the beta sheet. Normally composed of proline, glycine, alanine.
448
What base pair interactions promote RNA intra-strand interaction?
Non-trans-Watson Crick base pairing, i.e. not just binding at the Watson-crick edge, but also uses cis watson-crick base pairing, and bonding along the sugar edge and Hoogsteen edge of the nucleotide.
449
What does sulfation modification to protein tyrosine residues achieve functionally?
Proteolytic processing of some protein precursors Intracellular transport of some secretory proteins
450
What are the two isozymes in the brain in liver that catalyze the phosphorylation of glucose upon entering the cell, and why do we need two different isozymes?
The brain needs sugar to function more than the liver, so the Km of the phosphorylation reaction in the brain by hexokinase is much smaller (i.e. will catalyze reaction at much smaller concentrations of glucose). Glukokinase has a larger Km because entrapment of glucose in liver cells is less crucial and can tolerate waiting until glucose concentrations are higher.
451
Summarize effects of CD4 mimics, PT and PT thiols, and reversible conformational blockers on HIV-1 activity, and where on the gp120 protein these drugs bind. (Binding to different residues even in the same pocket causes drastic conformational and functional changes!)
CD4 mimetics just inactivate binding of p120 to CD4 by binding the Phe43 subcavity. Infection can still be activated. The mature, unliganded state of HIV-1 can still enter the prehairpin intermediate state by coreceptor binding and finally the post-fusion state by gp41 binding to the CCR5 protein on T cell membrane surface. Reversible conformational blockers also inhibit gp120 binding to CD4 by binding the gp120 beta 20/21 subcavity, preventing any type of binding by closing up the conformation, but can be reversed to allow HIV-1 infection. PT and PT thiols bind CD4 binding site of gp120 and trap the conformational state, inhibiting HIV-1 infection. PT's bind to both subcavities, and this is believed to be what triggers HIV-1 env inactivation.
452
Describe the subunits and binding regulation of ATCase.
ATCase has many subunits = trimer of regulatory dimers, and a dimer of catalytic trimers. This is called a supermolecular complex. When the product, CTP, binds the regulatory site, the T state is stabilized. When ATP, an activator, binds the regulatory site, teh R state is stabilized and the substrates can bind the catalytic sites. Here, flexibility is important as it allows for the conformational changes required for regulation by the allosteric regulators.
453
Describe phenylketonuria and the role played by enzyme defficiency.
Defect in enzyme that metabolizes phenylalanine to tyrosine, so tyrosine becomes an essential amino acid and the child cannot consume phenylalanine.
454
Which form of RNA has a complementary strand?
Only viral RNA.
455
define stereoisomer
same formula, different conformation.
456
Explain the process of N-glycosylation.
Dolichol pyrophosphate (lipid pointing to cytosol on membrane of ER) binds to a constant and complex, activated oligosaccharide. The phosphorylated lipid then flips to the inside of the ER. A nascent polypeptide that has the signal sequence for secretion or membrane relocation is extruded into the ER through its membrane where the entire oligosaccharide is transferred from the dolichol pyrophosphate to the nascent polypeptide. The glycoprotein then transits through the golgi where the oligosaccharide is modified. Glycosidases trim off sugars, and glycosyltransferases add sugars.
457
What enzyme type catalyzes formation of phosphodiester bonds?
DNA or RNA polymerases.
458
What is unusual about glycine?
It is unusually small and flexible.
459
What role does histidine F8 of hemoglobin play in binding of O2 to heme?
It forms the fifth coordination bond with iron in the heme group.
460
What is furanose?
461
How can RNA for ubstable and unique structures without concern for degradation?
The nucleotide sequence coding for the RNA molecules is store in DNA, which is much more stable.
462
Descibe substrate concentration as Vmax is approached asymptotically in a reaction following Michaelis-Menton kinetics.
Substrate concentration would be very high. Enzyme active sites will be saturated.
463
What is the second substrate of peptide hydrolysis by chymotrypsin?
It is water that helps the second product leave the acyl-enzyme.
464
What are 8 covalent post-translation modifications to proteins?
Proteolytic processing • Glycosylation • Lipid attachment • Sulfation • Carboxylation • Hydroxylation • Phosphorylation • Acetylation
465
