Kinetics and Regulation Flashcards
What is meant by reaction velocity?
it measures the how much reactant disappears as function of time or how much product appears.
V= -dt [A]/dt= dt [P]/dt a= substrate P= product. d=decrease or increase of substrate [ ].
What is enzyme kinetics?
studying the rate of enzyme-catalyzed reactions.
What does the Michaelis-Menten equation describe? Write the equation.
the initial reaction velocity as a function of substrate concentration.
(also determines variation of enzyme activity). V0= Vmax [S}/[S] + KM
Define what vmax and km are. What do they mean?
Vmax- maximum velocity possible, only achieved when all of the enzyme (ET) is bound to substrate. V= k2[E]T
Km- Michaelis constant that is the substrate concentration that yields 1/2 vmax.
Km is also total of constants: kM= k-1 + k2/k1; Km values for enzymes vary widely and is approximately substrate [ ] in vivo (living things).
What components make up Lineweaver- Burk plot? What are the variables for graph?
it is a double reciprocal plot that yields straight line equation:
composed of inverse of Vo as a function of inverse of substrate concentration
1/Vo= kM/Vmax times 1/S + 1/Vmax.
Slope= KM/Vmax; Y- intercept: 1/vmax ; x intercept: -1/Km
What is catalytic efficiency? What are the variables involved? What are the conditions for this to occur. How is the michaelis-menten equation adjusted for catalytic efficiency?
the efficiency of an enzyme in catalyzing a reaction.
Kcat/Km is a measure of catalytic efficiency, as it takes into account, both rate of catalysis (kcat) and nature of enzyme substrate interaction (Km)
stems from [S} being less than Km, since a lot of active sites are unoccupied and the free enzyme, [E}= E{T}
Michaelis equation will yield Vo= kcat/Km [S}{E]T
What are allosteric enzymes? How do they work?
enzymes that control the flux of biochemical reactions in metabolic pathways.
Due to their regulatory properties, they allow for generation of complex metabolic pathways.
Mechanism: activator or inhibitor molecule will bind to specific regulatory site on enzyme (not active site), which induces conformational or electrostatic changes to either enhance or reduce activity.
Distinguish between concerted model and sequential model of allosteric enzymes. Which molecule is a good example of this?
Concerted model - A conformational change in one subunit will be conferred to ALL other subunits (one r state, lead to all 4 being in r state)
Sequential model-subunits undergo sequential changes in structure.
when measuring the disappearance of A (substrate), what is the velocity of reaction? Write the equation and describe relationship between variables.
V= k [A], k is a proportionality constant.
The velocity of reaction is proportional to the reactant concentration.
What’s the difference between first order reaction? vs Second- order reaction? Unit difference?
First order reaction- The velocity is proportional to reactant concentration (as V increases, concentration of reactant gets higher) V=k [A ]. measured in s-1 units.
Second order reaction: bimolecular rxns where two reactants are involved. The rate of reaction is proportional to the product of the two reactants.
V= k [A}^2 AND V= k [A] [B}
proportionality constant k has M-1 s-1 units.
How is initial velocity (Vo) measured?
by measuring the product formation as a function of time, at beginning of reaction, and then determining velocity soon after reaction has started.
How should you evaluate enzyme kinetics? Why?
measure velocity as a function of substrate concentration with fixed amount of enzyme concentration. This is b/c enzyme [ ] is constant, while substrate concentration may vary depending on environmental conditions. this is when t= 0 and [P] = 0
E+S > ES > E + P ; there are rate constants on arrows in equation. k1 is for first step E and S form ES complex (k-1 is the reverse reaction of this) k2 is the second step ES to E + P(k-2 is reverse rxn, but it is ignored by keeping [P] 0. )
When viewing velocity vs substrate concentration graph, why does the curve eventually level off?
it levels off since the enzyme is saturated and V max has been reached.
What are the two fates of the ES complex?
ES can proceed to form Product P with rate constant k2, or go backwards and dissociate into E + S, k-1.
What is Vmax directly dependent on? independent? what happens at high substrate concentrations, when [S] is higher than Km?
depends on enzyme concentration.
Vmax independent of substrate concentration
if [S} is higher than Km, Vo= Vmax
What occurs when vmax is reached?
When vmax is reached, the total amount of enzyme is bound to substrate, enzyme is saturated. Also adding more substrate will not affect velocity, since it’s already at its max.
When does Km approximate dissociation constant of ES complex?
When k-1 (reverse step) is greater than k2 (forward, form product)
what does a high Km indicate in terms of binding? Low Km?
High Km- weak binding
low Km- strong binding
What is the steady state?
the assumption that concentration of reaction intermediates remain constant throughout reactions (they’re being produced as being consumed).
Which two enzymes play a key role in metabolism of alcohol? What are the reactants and products in the equation.
Alcohol dehydrogenase and Aldehyde dehydrogenase.
Ch3ch2OH (ethanol) + NAD+ using alcohol dehy. forms CH3CHO + NADH and H+
CH3CHO + NAD+ + H20 using aldehyde dehy. forms CH3COO- (acetate) + NADH + 2H+
Explain what high km and lowe km mean in terms of catalysis and substrate concentration.
With low km, their is a high affinity, and more catalysis occurs at lower substrate concentrations
High km- lower affinity; less catalysis at lower substrate concentration.
How does Km levels affect how people respond to alcohol consumption?
After drinking alcohol, some people may have symptoms of facial flushing, rapid heart beat due to excessive amounts of acetaldehyde in the blood. 2 different forms of acetaldehyde dehydrogenases in people.
Those individuals susceptible to symptoms have inactivated low km. They instead have HIGH km, and cannot process all of acetaldehyde (weak binding) , some acetaldehyde appears in blood, causing symptoms.
What is one enzyme/Substrate pair that has high affinity of binding? Low affinity?
High affinity (due to low km value)- E: Lysozyme and S: Hexa-N-acetlyglucosamine Low affinity (high km)- E: carbonic anhydrase and S: CO2.
Write an equation for when the total enzyme concentration, [E] T is known.
Vmax= k2[E] T or k2= Vmax/[E}T
What is Kcat? How does it relate to k2?
Kcat- is the turnover number of enzyme, or number of substrate molecules that an enzyme can convert into products per second. Kcat is also the same as k2, as the turnover rate is = to constant (k2).
What is the relationship between kcat and turnover?
The higher the kcat value, the more substrates are turned over (converted into product)
Which enzyme has the highest turnover numbers? lowest?
highest turnover numbers- carbonic anhydrase.
lowest turnover numbers- lysozyme
hence enzymes with higher binding affinities (low km), have lower turnover numbers.
What other information can can kcat/Km (catalytic efficiency) provide?
can provide info about the preferences enzymes have for different substances based on kcat/km value.
Ex: Chymotrypsin has a preference for bulky hydrophobic side chains (phenylalanine, norvaline/norleucine.
What are the two main groups of multiple substrate reactions? distinguish between the two and describe the different mechanisms.
- Sequential reactions- form ternary complex consisting of two substrates and enzyme.
Mech: the first substrate(NADH) will bind to enzyme, followed by second substrate (Pyruvate)to form complex. all substrates must first bind to the enzyme, before being catalyzed and released as products (lactate and NAD+) - Double-displacement reactions (ping pong)- characterized by formation of substituted enzyme intermediate.
Mech: first substrate (aspartate) will bind to enzyme, then catalytic step occurs, which creates substituted enzyme (E-NH3), and substrate is released as product (oxaloacetate); then second substrate(alpha ketoglutarate) will bind to substituted enzyme, creating catalytic step and NH3 transfers to substrate form last product (glutamate).
Hence this is like ping-pong where substrates and products bounce on and off.
What kind of curves do allosteric enzymes and Michaelis menten enzymes have on graph and why? what is the physiological significance of Allosteric curve over Michaelis Menten?
Michaelis menten- hyperbolic curve
allosteric enzymes- sigmoidal curve (due to sharp increase in Vo at middle of graph)
significance of sigmoidal curve for allosteric enzymes: they can transition from a less active state to a more active state with a narrower range of substrate concentration (change rate quicker with smaller substrate [} )
Micahelis (requires 27 fold increase in [ S] ), while Allosteric needs 4 fold increase in [ S]
Which confirmation state of allosteric enzymes is more stable, T or R state? How does this affect binding affnity?
R (relaxed is the more stable state, as it is easier for substrate to bind to enzyme, and requires less [S} T state (Tense) is less stable, more difficult for substrate binding to enzyme. Requires more [S]
Explain what the the T and R state represents as well as describe the shape each state has.
T- Tense state ; square shape
R- Relaxed state: circle shape
What is the threshold state? How does it work?
describes the sensitivity of allosteric enzymes, and how they are more sensitive to changes in substrate concentration near Km than Michaelis Menten.
below a certain substrate [ ], there is little enzyme activity. After threshold is reached, enzyme activity increases rapidly.
What is cooperativity? How does it affect the allosteric enzyme curve and work like a switch?
behavior in which the initial binding of substrate to one active site in R form or any form will lead to other active sites being trapped in R (or other state). binding of substrate will disrupt T to R equilibrium in favor of R (make it easier for substrate to bind R)
cooperativity is what causes sharp increase in Vo for Velocity vs substrate concentration. It works like an on/off switch: either the majority of enzyme is ON state (R state) or OFF state (T state)
In a metabolic pathway with 5 reactions from A to F, what is the committed step and how does it affect rest of reactions? which enzyme catalyzes the committed step? Which does the remaining steps?
The committed step is the conversion of A to B, and after this step occurs, B will be committed to being converted to F (all of the remaining steps will continue).
Allosteric enzymes catalyze committed step of metabolic pathways
Michaelis- Menten enzymes facilitate the remaining steps.
Distinguish between the features for michaelis menten and allosteric enzymes.
Michaelis menten- enzyme velocity only depends on substrate concentration and S forming products; only catalysis role.
Allosteric enzymes- regulate metabolic pathways, series of roles including regulating inhibitory and stimulatory molecules, receiving and transducing chemical signals
What is feedback inhibition? How does this apply to 5 step reaction A to F? How does it work?
way of regulating biochemical reactions by preventing the production of too much product.
Feedback inhibition helps regulate the amount of F synthesized (prevent it from making too much of F).
Mech: When enough F is present, F will bind reversibly to e1 which is enzyme used for committed step of reaction (A to B) and will inhibit reaction.
The pathway product F inhibits enzyme e1 by binding to regulatory site of enzyme distinct from active site.
Why can the regulation of metabolic pathways be quite complex? What is the structure of allosteric enzymes?
because allosteric enzymes may be inhibited or stimulated by several regulatory molecules.
What makes Allosteric enzymes so special?
They DO NOT conform to Michaelis Menten kinetics; They have unique features of how they respond to changes in substrate concentration and also use sigmoidal kinetics.
Structure- 4 subunits on 4 active sites.
Describe the importance of quaternary structure in allosteric enzymes.
Allosteric enzymes depend on alterations in quaternary structure that has multiple active sites, regulatory sites.
binding of one substrate to one active site, will trap the other active sites in R state and remove substrate bound enzyme from T to R equilibrium.
Disruption of T- R equilibrium by binding of substrate favors conversion of more enzymes to R state.
What are the features of the concerted model?
- The enzyme exists in 2 different quaternary structures designated T (tense state) or R (relaxed state).
- If T and R are in equilibrium (no signals or substrate), T will be more stable state.
- The R state is enzymatically more active (will catalyze reactions) than T state
- all active states must be in same state (ALL or NONE; either all R or all T) (aka symmetry rule)
also substrate binds more readily to R form of enzyme than T form.
What is L0?
the allosteric constant, where L0= T/R ratio
What is the purpose of regulatory molecules in allosteric regulation? compare and contrast the two regulator molecules? How do they affect threshold concentration?
allosteric regulators disrupt the R to T equilibrium when they bind the enzyme.
Activators, or positive effectors bind to R form at regulatory site of enzyme to stabilize R form( thus increasing concentration of R, make R and S interaction more likely).
Inhibitors or negative effectors, binds to T to stabilize T form (thus increasing T [ ] and reducing likelihood of R and S interaction).
Activators will lower threshold [ ] needed for enzyme activity; inhibitors raise threshold [ ]
Distinguish between homotropic effect and heterotrophic effect. How does each kind of effect influence graph curves/ What makes homotropic so special?
Hetertropic effect: the effect of REGULATORY molecules on allosteric enzymes;
for Hetero effect, the sigmoidal curve will shift left ( activators, ATP) and right( inhibitors, CTP)
Homotropic effect; the effect of SUBSTRATES on allosteric enzymes. This homotropic effect is used mainly for sigmoidal nature of the kinetics curve (black curve)
