Conceptual Exam 2 Flashcards
Kd equation in terms of vmax and concentration?
Low kD equals?
high affinity
Free Energy equation?
Function of myoglobin?
Carries oxygen in muscle cells; binds oxygen very tightly
What is p50?
The partial pressure of O_2 that results in 50% of the binding sited of myoglobin being occupied.
How many subunits does myoglobin have?
One subunit
Cooperative or uncooperative binding in myoglobin?
Uncooperative
What is the function of the distal His?
stabilizes O_2 (donates proton)
Does O_2 or CO bind better? Why?
The CO bond is a triple bond which is stronger than O_2’s double bond. Greater polarity.
What is the makeup of hemoglobin?
single domain heterotetramer (4 subunits to bind to O_2; two alpha, two beta)
Describe T-state’s affinity and kD for O_2?
High kD, low affinity
Is hemoglobin binding cooperative or uncooperative?
Positively cooperative
What does cooperativity mean?
binding to one heme group raises the affinity of other subunits for oxygen
Draw myoglobin’s binding graph.
What does binding induce?
A conformational; change
Deoxyhemoglobin O_2 or none? Oxyhemoglobin?
None bound; O_2 bound
Describe R-state’s affinity and kD for O_2?
Low kD, high affinity
What is the O_2 binding curve? Draw it.
a visual representation of hemoglobin saturation
What does the transition involve from T-state to R-state?
broken ionic/electrostatic interactions
What is a cofactor? Function?
One or more inorganic ions or complex molecules called coenzymes; helper molecules for enzymes to assist them with biological functions
What is an enzyme? What does it lower?
a biomolecule (protein or RNA) that catalyzes a chemical reaction
What is a prosthetic group?
A coenzyme or metal ion that is tightly bound to enzyme
Draw out the reaction coordinate diagram.
What is an apoenzyme? Holoenzyme?
enzyme without its cofactors; a complete catalytically active enzyme together with its bound cofactors
What is an active site?
pocket within enzyme at which substrate binds
What is the substrate?
Compound that is acted upon by enzyme
Free GIbbs Energy equation for Keq?
What do enzymes do and what is the end result?
lower activation energy, making it faster for rxn to occur
Keq is equal to?
products/reactants
Relationship of enzymes and transition state? What does this mean?
Complementary, active site of enzyme is tailored to stabilize the transition state of the reaction
What remains unchanged in the reaction coordinate diagram?
change in G, no altered equilibrium
What do transition state analogs do? What do they compete with?
They are molecules designed to mimic the transition state during a reaction; they compete with the substrate for binding at the enzyme’s active site
What are the 5 rate enhancement rules?
- specificity
- covalent catalysis
- acid-base catalysis
- proximity/orientation
- transition state stabilization
Explain acid-base catalysis.
Acid or base (other than H+ or OH- that accelerates the reaction)
When is the proton transferred in acid-base catalysis?
transition state
Explain covalent catalysis.
Characterized by the formation of a covalent bond b/w enzyme and substrate
Good acids/bases?
Glu, Asp, Lys, Arg, Cys, His, Ser, Thr, Tyr
What does chymotrypsin use?
acid/base and covalent catalysis
Residues that participate in chymotrypsin and their role?
1) Ser 195- nucleophile in active site
2) His 57- base, accepts proton from Ser 195
3) Asp 102- acid, donated proton to amino group and promotes departure
What is the experimental determination of enzyme kinetics?
measure reaction rates of enzyme under different substrate concentrations
What is Michaelis-Menten Equation? identify the components.
Draw the Lineweaver-Burk graph.
Draw the Michaelis- Menten equation.
What is the Lineweaver-Burk Equation?
What is Km the rate of?
breakdown of ES/rate of formation of ES
Why are these assumptions helpful?
They simplify the mathematics.
What are the two assumptions for M-M?
- Conc of enzyme-substrate complex remains constant over time (steady state/equilibrium)
- Reaction is observed at initial phase, where substrate conc is much higher than enzyme conc (initial velocity)
What is the turnover number (Kcat)?
number of substrate molecules converted to product per enzyme molecule per unit time
Identify the equations that differentiate Km from Kd? When are they equivalent?
——–, when K_-1 is»_space;»> K_2
Draw out the aspartic proteases.
What is catalytic efficiency?
effectiveness of an enzyme in facilitating a reaction
What is the Kcat equation?
What is diffusion in limited catalysis?
Enzyme’s catalytic rate approaches max diffusion rate of substrate to enzyme’s active site
What is the equation for catalytic efficiency?
Kcat/Km; cat/mouse
In competitive inhibition, where does the inhibitor bind? Km affect? Vm?
active site, Km increases, Vm doesn’t change
Draw out a pH activity curve.
Draw out M-M and Lineweaver graphs for competitive inhibited and uninhibited enzymes.
Draw out M-M and Lineweaver graphs for uncompetitive inhibited and uninhibited enzymes.
Where does mixed inhibition bind? Effect of Km and Vmax?
binds at site distinct from substrate binding site; can increase or decreae
Describe irreversible inhibition. Example?
combine with or destroy functional group on enzyme that is essential for activity; penicillin
What is the catalytic triad?
His 57, Ser 195, Asp 102
What do serine proteases do (2)? What do they contain?
- cleave peptide bonds
- conserve 3 degrees structures
Contains catalytic triad
Draw out the arrow pushing mechanism for serine protease.
What does change in color intensity indicate?
Changes in absorbance at specific wavelengths can reveal conc of substrate/product over time
What does a calorimetric substrate/product allow for?
Measurement of enzymatic activity based on changes in color intensity
Why is nitrophenolate a useful probe?
You can mix with enzyme and watch immediate cleavage reaction.
Nitrophenolate mechanism.
What does Km vs. pH reveal?
pH the binding affinity is highest or lowest
What is burst kinetics? Draw graph.
Initial rapid burst of product formation followed by a slower, steady-state phase; ————
What does a crystal structure allow for?
Allows visualization of the active site
What do pH and activity profiles show?
Shows how enzyme activity varies with changing pH levels
How are AP and Chymotrypsin mechs different and similar?
-different active site residues
-AP used nucleophilic attack of H_2O, chym. uses nucleophilic attack of serine residues
-both enzymes exhibit specificity for cleaving peptide bonds
-both form covalent enzyme-substrate intermediate during catalysis
What does Kcat vs. pH reveal?
pH at which enzyme achieves max catalytic efficiency
high Km equals? low Km?
low affinity, high affinity
Draw out enolase mechanism.
What is the purpose of enzyme regulation?
respond to changing needs of the cell
What is allosteric regulation?
molecule binds to allosteric site and causes conformational change that alters enzyme’s activity
What is feedback inhibition?
end product of metabolic pathway binds to proton inhibits an enzyme earlier in the pathway
What is covalent modification?
phosphorylation, acetylation, methylation —> alters enzyme activity, stability
What element does enolase use? What does it do?
Mg2+, stabilizes negative charge
What is proteolytic cleavage?
some enzymes are inactive and require proteolytic cleavage to become active
Draw out the kinetic behavior of allosteric enzyme. Pos and neg.
What does the hill coefficient measure?
cooperativity
If N>1, pos or neg cooperativity? End result meaning?
positive, means that it enhances binding
If n <1, pos or neg? Meaning?
Negative, inhibits binding of subsequent molecules
Why is the coefficient useful?
influences enzyme’s sensitivity to changes in substrate concentration
What is feedback inhibition?
feedback mechanism to respond to needs of isoleucine
What is covalent modification?
Phosphorylation (adds phosphate, makes molecule bigger and more negative)
What adds phosphate? Removes it?
Kinase and phosphatase
Mechanism of phosphorylation.
Draw out the proteolytic cascades for chymotrypsin maturation/blood clotting.
