MSK - Biochemistry - Enzymes; Microtubules Flashcards
What are enzymes?
How do they accomplish their function?
Biological catalysts (mostly proteins, but some are RNA);
by lowering activation energy
Do enzymes change the overall thermodynamics of a reaction?
(I.e., can they change equilibrium states?)
No; equilibrium is not changed but the reactions reach equilibrium more quickly
Under what conditions do human enzymes operate?
Very mild conditions
(aqueous environment, fairly neutral pH, body temperature)
How many types of reaction can a single enzyme produce?
With how many substrates can a single enzyme produce its specified product?
One
(reaction specificity);
one, or one class of structurally similar substrates
(substrate specificity)
What type of chemical interactions do enzymes use to bring substrates together in favorable ways towards the product of the reaction?
Weak bonds (I.e., not covalent bonds)
these can be hydrogen bonds, hydrophobic bonds, ionic bonds, or van der Waals (London forces) interactions
If a reaction is thermodynamically stable, the product will always have a ________ ground state than the substrate.
Lower
What is the name of the peak point of the activation energy for a particular reaction?
Transition state energy
What is ΔG in terms of enzymatic reactions and activation energy?
The change in free energy
(i.e. the energy required to reach the transition state energy, aka the peak of the activation energy)
True/False.
Enzymes lower ΔG.
True.
What is ΔG’°?
A favorable reaction will have a __ ΔG’°.
An unfavorable reaction will have a __ ΔG’°.
The total change in free energy for a reaction
(from the transition state energy [peak activation energy] to the product energy);
-
+
Why are transition state analogues useful in binding enzymes?
They tend to bind more forcefully than substrate or product analogues would
What type of analogue tends to bind enzymes more forcefully than either substrate or product analogues?
What are two examples of this?
Transition state analogue;
most HIV protease inhibitors,
Oseltamivir (TAMIFLU) - binds neuraminidase
What enzymatic class transfers electrons from donors to acceptors?
Oxidoreductases
Redox reactions mostly involve what atoms (elemental types) in our cells?
Carbon, nitrogen, sulfur
What two mnemonics can be used to remember which molecule is reduced and which is oxidized in a reaction based on the movement of electrons?
LEO the lion says GER;
OIL RIG
What is velocity in terms of enzyme kinetics?
The rate of appearance of P (product)
What is initial velocity (vi or v<span>0</span>) in terms of enzyme kinetics?
The rate of product appearance at t = 0
(before any loss of substrate or other change in conditions)
(basically, peak velocity for the enzyme in question)
True/False.
Initial velocity (vi or v0) will remain unchanged even if enzyme concentration increases.
False.
Left graph: different initial velocities (dashed line) for different enzyme concentrations
Right graph: linear relationship between initial velocity and enzyme concentration
True/False.
A high concentration of enzyme will reach reaction equillibrium faster than a low concentration of enzyme?
True.
The Y-axis of a Michealis-Menten graph shows:
The X-axis of a Michealis-Menten graph shows:
Velocity (rate of product formation; often mmoles/sec)
Substrate concentration [S]
In Michaelis-Menten enzyme kinetics, the value for Km is __ Vmax.
1/2
Km is:
Vmax is:
the substrate concentration [S] at 1/2 Vmax
the velocity at an infinte amount of [S]
State the Michaelis-Menten equation.
What is the general hypothesis of enzyme kinetics on which the Michaelis-Menten equation builds?
E + S -> ES* -> E + P
OR
E + S <– ES
At infinitely large [S], V0 = ?
At small [S], V0 = ___________ proportional to ____
Vmax;
linearly, [S]
Will enzymes with a high affinity for a particular substrate have a low or high Km?
Low
A high Km indicates what relationship between enzyme and substrate?
Low affinity
True/False.
Km is the velocity at half of Vmax.
False.
Km is the substrate concentration at Vmax/2
Hexokinase I has a much lower Km [glucose] than glucokinase (hexokinase IV).
Which has a lower affinity for glucose?
Glucokinase (hexokinase IV)
The Km values for Hexokinase for Glucose and fructose are Km = 0.05 mM and Km = 1.5 mM, respectively.
Hexokinase has a higher affinity for which substrate?
Glucose
What is Kcat in terms of enzyme kinetics?
How is it expressed?
The speed one enzyme has in turning out product when saturated with substrate;
products / sec
What are the four main types of enzyme inhibition?
(Note: one of these types is a subset of another)
Competitive
Mixed (subset: noncompetitive)
Uncompetitive
Noncompetitive inhibitors are a subset of ___________ inhibitors.
Mixed
What type of inhibitor is shown in this Lineweaver-Burke plot?
Competitive
What type of inhibitor is shown in this Lineweaver-Burke plot?
Mixed
What type of inhibitor is shown in this Lineweaver-Burke plot?
Noncompetitive
What type of inhibitor is shown in this Lineweaver-Burke plot?
Uncompetitive
What formula works out the Y = mX + b for Lineweaver-Burke plots?
1/V0 = (Km/Vmax) * (1/[S]) + (1/Vmax)
What is the Y in Y=mX+b for a Lineweaver-Burke plot?
1 / V0
What is the m in Y=mX+b for a Lineweaver-Burke plot?
(Km / Vmax)
What is the X in Y=mX+b for a Lineweaver-Burke plot?
(1 / [S])
What is the b in Y=mX+b for a Lineweaver-Burke plot?
(1 / Vmax)
(the Y-intercept)
The Y coordinate for a Lineweaver-Burke plot is:
The X coordinate for a Lineweaver-Burke plot is:
1 / V0
1 / [S]
The slope for a Lineweaver-Burke plot is:
The Y-intercept (b) for a Lineweaver-Burke plot is:
The X-intercept for a Lineweaver-Burke plot is:
Km / Vmax
1 / Vmax
- 1 / Km
The X-intercept for a Lineweaver-Burke plot is:
The Y-intercept for a Lineweaver-Burke plot is:
- 1 / Km
1 / Vmax
Describe competitive inhibition effects in regards to the following:
Km
Vmax
Binding location on the enzyme
Can it be overcome by increasing substrate concentration?
Increase;
no change;
at the active site;
yes
Describe mixed inhibition effects in regards to the following:
Km
Vmax
Binding location on the enzyme
Can it be overcome by increasing substrate concentration?
Increase;
decrease;
distant from the active site (binds either E or ES)
no
Describe noncompetitive inhibition effects in regards to the following:
Km
Vmax
Binding location on the enzyme
Can it be overcome by increasing substrate concentration?
No change;
decrease;
distant to active site;
no
Describe uncompetitive inhibition effects in regards to the following:
Km
Vmax
Binding location on the enzyme
Can it be overcome by increasing substrate concentration?
Decrease;
decrease;
distant to active site (only to ES);
no
Describe the effect of each of the following types of inhibitor on Km:
Competitive
Mixed
Noncompetitive
Uncompetititve
Increase
increase
No change
Decrease
Describe the effect of each of the following types of inhibitor on Vmax:
Competitive
Mixed
Noncompetitive
Uncompetititve
No change;
decrease;
decrease;
decrease
Describe to what each of the following inhibitors binds (e.g. E, ES, etc.):
Competitive
Mixed
Noncompetitive
Uncompetititve
E
E, ES
E, ES
ES
What type(s) of inhibitor can bind either the enzyme (E) or the enzyme-substrate (ES) complex?
Mixed
(and noncompetitive, a subtype of mixed)
What type(s) of inhibitor can be overcome by increasing substrate concentration?
Competitive only
What type of inhibitor can only bind the enzyme-substrate (ES) complex?
Uncompetitive
What type(s) of inhibitor bind(s) enzymes distant to the active site?
Mixed (and the noncompetitive subtype);
uncompetitive
Describe the Lineweaver-Burke plot of an enzyme with uncompetitive inhibition (especially in regards to Km and Vmax).
Decrease in both
Describe the Lineweaver-Burke plot of an enzyme with noncompetitive (a type of mixed) inhibition (especially in regards to Km and Vmax).
Decrease in Km;
no change in Vmax
Describe the Lineweaver-Burke plot of an enzyme with competitive inhibition (especially in regards to Km and Vmax).
Increase in Km;
no change in Vmax
Describe the Lineweaver-Burke plot of an enzyme with mixed inhibition (especially in regards to Km and Vmax).
Increase in Km;
decrease in Vmax
What is the most common type of enzyme inhibitor in the body?
How does it typically work?
Mixed;
allosteric regulation
Describe allosteric inhibition.
A ligand binds an enzyme (distant to its active site) and decreases its affinity for its substrate
What type of inhibition occurs when a ligand binds an enzyme (distant to its active site) and decreases its affinity for its substrate?
Allosteric regulation
A product of glycolysis inhibits (provides negative feedback to) an early enzyme of glycolysis.
This is an example of what type of inhibition?
Allosteric regulation
How do irreversible inhibitors work?
They covalently bind active sites
What are mechanism-based (suicide) inhibitors?
What is an example class?
(Name two examples of this class.)
Irreversible inhibitors (they covalently bind active sites) that are activated by the enzyme they inhibit;
serpins
(α 1-antitrypsin, antithrombin III)
What type of inhibitor is here described:
an irreversible enzyme that is activated by the enzyme it inhibits
What is are two examples?
Mechanism-based (suicide) inhibitors;
α 1-antitrypsin, antithrombin III
(both serpins)
True/False.
Allosteric regulation can be used to both increase and decrease enzymatic activity.
True.
Hemoglobin is an example of an allosterically regulated protein that shows a ___________ curve when plotted against [S].
True/False.
Many allosterically regulated enzymes show this curve.
Sigmoidal;
true
When substrate concentration is much greater than Km, this is a ____ order reaction.
Zero
When substrate concentration is less than Km, this is a ____ order reaction.
First
When [S] = Km, V0 =
1/2 Vmax
Enzymes stabilize what to decrease activation energy?
The transition state
What molecule is bound to both α- and β-tubulin monomers?
GTP
The GTP attached to which tubulin monomer in microtubules becomes hydrolyzed over time and has effects on microtubule stability?
β-tubulin
How does taxol exert an effect on cancer cells?
It stabilizes microtubules, halting mitosis
Which types of cytoskeleton are polarized?
Microfilaments;
microtubules
Which tubulin monomer lines the positive end of protofilaments in microtubules?
β
Which tubulin monomer lines the negative end of protofilaments in microtubules?
α
Which ring structure is found in microtubules during interphase or mitosis?
Singlets
Which ring structure is found in microtubules in cilia and flagella?
Doublets
Which ring structure is found in microtubules in basal bodies and centrioles?
Triplets
What forms at low concentrations of α- and β-tubulin monomers?
Tubulin dimers
After reaching critical concentration of α- and β-tubulin, what occurs?
The forming dimers polymerize into microtubules and the dimers concentration no longer increases
Which end of the microtubule polymerizes most rapidly?
Which end of the microtubule depolymerizes most rapidly?
The + end;
the + end
Which end of the microtubule has a lower critical concentration [dimers] needed in order to stimulate polymerization?
The + end
Which end of the microtubule protofilament is least stable?
Why?
The - end;
due to treadmilling (older monomers with more hydrolyzed GTP are found at the - end)
Why is the process of treadmilling important to microtubule protofilaments?
Older β-tubulin monomers are sent towards the back
(higher concentrations of GDP = less stability)
What provides an especially important aspect of stability to the + end of microtubules?
GTP caps
Dimers can only be added to the + end of microtubules if the ____ ____ is present.
GTP cap
If the tubulin dimer pool is greater than the critical concentration, then what occurs?
Microtubules polymerize, faster at the + end
What is microtubule treadmilling?
Polymerization at the + end & depolymerization at the - end
Name three microtubule associated (stabilizing) proteins (MAPs).
Tau,
MAP2,
MAP4
Name three microtubule destabilizing proteins.
Kinesin-13,
stathmin,
katanin
The positively charged groups in microtubule associated proteins (MAPs) bind to negatively charged groups in microtubules. What effect does this have?
How are these proteins inactivated?
Microtubule stabilization
(due to charge neutralization);
phosphorylation
Via what two methods do microtubule destabilizing proteins destabilize microtubules?
Bending microtubule protofilaments;
hydrolyzing GTP
What happens to Tau protein in order for tau aggregations (neurofibrillary tangles) to form?
What is Tau’s normal function?
Hyperphosphorylation;
microtubule stabilization
Both amyloid precursor protein and Tau protein contain a phosphoThreonine–Proline motif. If the proline is in the ___-configuration, then phosphorylation is increased, increasing risk of Alzheimer’s disease.
What protein helps to switch this ___-proline back to the ___ -configuration and is protective against AD?
Cis;
cis, trans; proline isomerase (PIN1)
Kinesin binds to which tubulin monomer of microtubules?
β-tubulin
Which type of motor protein ‘walks’ along microtubules with two foot-like projections?
Which type of motor protein ‘hops’ along microtubules with a single stalk?
Kinesin;
dynein
What molecule is hydrolyzed to allow for kinesin ‘walking’ and dynein stalk power strokes?
ATP
When does the centrosome (MTOC) replicate?
When do the two spindle poles migrate to either end of the cell?
In late interphase (just before mitosis);
prophase
What are the three types of microtubule present in the spindle apparatus?
Aster,
kinetochore,
polar
What protein holds sister chromatids together and must dissolve during anaphase?
Cohesin
What are aster microtubules?
Extend from the spindle poles towards the cell cortex and orient the spindle poles with respect to the axis of cell division
What type of mitotic microtubule is here described?
Extend from the spindle poles towards the cell cortex and orient the spindle poles with respect to the axis of cell division
Aster microtubules
What do kinetochore microtubules do?
Bind the kinetochore of the chromosome centromere and pull apart the sister chromatids during early anaphase
What type of mitotic microtubule is here described?
Bind the kinetochore of the chromosome centromere and pull apart the sister chromatids during early anaphase
Kinetochore microtubules
What are the three main functions of polar microtubules?
- Push duplicated centrosomes apart in Prometaphase
- Help orient the central spindle
- Push the spindle poles further apart in late anaphase
What type of mitotic microtubule is here described?
- Push duplicated centrosomes apart in Prometaphase*
- Help orient the central spindle*
- Push the spindle poles further apart in late anaphase*
Polar microtubules
What protein walks down aster microtubules, pulling the spindle apparatus towards the cell cortex?
What protein walks down polar microtubules, pushing the spindle apparatus apart?
Aster dynein;
kinesin
What is the cell cortex?
The actin layer covering the P-face of the plasma membrane
When do kinetochore microtubules bind to kinetochores?
Prometaphase
During mitosis, as the chromosomes are pulled / pushed into the metaphase plate, what protein connects kinetochore microtubules to kinetochores so that the end of the microtubule is free to polymerize?
Which depolymerizes the microtubule on the opposite side of the sister chromatids?
Kinesin-7;
kinesin-13
What amino acid on Tau protein can occur in the cis- or trans-conformation and is relevant to Tau tangle formation?
Which form is normal?
Which form leads to tangles?
What enzyme tries to correct the issue?
Proline (part of a phosphothreonine-proline motif);
trans;
cis;
proline isomerase 1 (PIN1)
How are the spindle apparatuses pulled apart during mitosis?
Dyneins attach to the cortex and motor towards the (-) end of aster MTs
(green circle in image)
What parts of kinesin attach to beta-tubulin?
The motor heads
What part of dynein performs ATP hydroylsis?
The head
When both kinesin heads are bound to tubulin, what happens so the posterior head can diassociate?
ATP hydrolysis
What happens in order for the unbound kinesin head to ‘step forward?’
ATP binding of the head bound to tubulin (the front head)
(NOT hydrolysis)
What region of a transport kinesin interacts with the microtubule?
What region of a transport kinesin interacts with the cargo?
The head domains;
the tail domain
Which kinesin head (front or back) does ATP bind in order to cause the ‘swing’ or ‘step’ forward of the back kinesin head?
The front head
Formin stimulates the formation of unbranched, __________ actin rings.
Contractile
True/False.
A defect in spectrin can result in small and fragile red blood cells.
True.
How does vitamin C help in collagen synthesis in terms of iron control?
It maintains iron in the Fe3+ form (oxidized)
What happens during anaphase A?
What happens during anaphase B?
Chromosomes are pulled apart (kinetochore microtubules shorten);
the spindle apparatuses are pulled apart (kinesins travel down polar microtubules)
