Enzyme Kinetics Flashcards
What are biological catalysts?
Enzymes
Except for some RNA or RNA/protein complexes (Ribozymes) most enzymes are what?
Proteins
The name of an enzyme usually ends with what?
-ase
What does the name of an enzyme describe?
Its function
What does sucrase do?
breaks down sugars
What do proteases do?
break down proteins
What do lipases do?
break down lipids
What does DNA polymerase do?
builds DNA
What do oxidases do?
catalyze oxidation reactions
Sometimes common names are used particularly for which enzymes
digestion
Most names describe the substrate and the what?
function (alcohol dehydrogenase)
What is a molecule that enzymes work on called?
substrate
What do we call the molecule that is produced by an enzymatic reaction?
Product
What is the part of the enzyme where the substrate binds and a reaction takes place?
Active-site
Enzymes are not what during the reaction
changed
What changes during a catalyzed reaction?
the activation energy
What do catalysts do to the rate of a specific reaction?
accelerate
What don’t catalysts do?
Alter the reaction (change thermodynamic) Get consumed (remain the same/regenerated)
Catalysts affect the rate of the forward and reverse reactions by what?
the same factor
Do catalysts affect the position of equilibrium?
No
What do catalysts lower for a reaction?
the activation energy required
What is the Arrhenius equation?
k=Ae^(-Ea/RT)
In the Arrhenius equation, what is A?
Frequency factor (frequency of collision)
What is activation energy?
The minimum energy required to start a reaction (to reach the transition state from the reactants)
What is the region within an enzyme that binds substrate molecules
Active site
What are the two proposed models of Enzyme-substrate interaction?
Lock-and-Key
Induced Fit Model
With Lock-and-Key, the active site has what kind of shape?
rigid
The specificity of enzymes can be explained by which model, but does not work for all enzymes?
Lock-and-Key
With Induced Fit Model, the active site is what shape?
flexible (the shapes of the enzyme, active site, and substrate adjust to maximize the fit, which improves catalysis) (greater range of substrate specificity)
Substrates are held in active site by weak interactions such as what?
hydrogen bonds and hydrophobic interactions
What does lowering the activation energy do?
- Stabilizes the transition state
- Provides a favorable microenvironment
- participation directly in the catalytic reaction
- speeds up the reaction
What affects the enzyme activity?
- Environmental Conditions (Temperature, pH)
- Cofactors and Coenzymes
- Enzyme Inhibitors
Using the optimum temperature allows for what?
greatest number of collisions between enzyme and substrate.
too high (denature protein-unfolded) too low (molecules move slower-fewer collisions)
pH affects what?
protein structure (activity)
What are two types of cofactors?
metal ions (inorganic) Zn Fe Mg Mn Cu Co small organic molecules (coenzymes)
Are cofactors proteins?
no
What is a non-protein component that is loosely bound to the apoenzyme by non-covalent bonds?
coenzyme (PLP, FMN, FAD, NAD+)
What is a non-protein component that is tightly bound to the apoenzyme by covalent bonds?
prosthetic group (Heme)
What do we call an enzyme without its cofactor or ligands?
Apoenzyme (inactive)
What do we call an enzyme that is active with its non protein component?
Holoenzyme
As the substrate concentration increases, the rate of the reaction does what?
increases
when does the maximum activity occur?
enzymes are saturated
What bring reactants to close proximity and align them in the proper orientation fro the reaction to happen via lowering the reaction activation energy?
Enzymes
Are enzymes reusable?
Yes
What is the change in concentration of a reactant/product divided by the time interval during which this change is observed called?
speed/rate of a chemical reaction
What sign is placed in front of the definition for reactants?
negative (being consumed)
what is the reaction rate of A + B -> C + D
rate = (-delta A/delta t)= (-delta B/delta t)=(delta C/delta t)=(delta D/delta t)
If there is a coefficient in front of a compound where does it show up in the rate equation?
the denominator
What is determined by taking the slope of a line tangent to the curve at that particular point?
instantaneous rate (first derivative of the function)
What is the change of concentration over a period of time?
Average rate
The larger the time interval used to find the average rate, the more the what?
deviation from the instantaneous rate
To measure the reaction rate, what do you need?
the ability to measure the concentration of at least one component in the mixture at different points in time
What often depends on the concentration of one or more of the reactants molecules?
the rate
The equation relating the concentrations of reactants to rate is called what?
The Rate Law
What would be the Rate Law of A -> Products?
rate = k[A]^n
k is the rate constant
n is the order (determines the rate dependence on reactant concentration)
The sum of the exponents on the reactants is called the what?
order of the reaction
What is the order of the following reaction: k[NO]^2[O2]?
3
If a reaction is zero order the rate of the reaction is what?
always the same rate = k[A]^0 = k
If a reaction is first order, the rate is directly proportional to what?
The reactant concentration, rate = k[A]
If a reaction is second order, the rate is directly proportional to what?
the square of the reactant concentration rate = k[A]^2
Doubling [A] will quadruple the rate of the reaction.
What must be determined experimentally?
The rate law
What method is the data from different experiments with varying starting concentrations of reactants used?
method of initial rates
What can be determined if you change the initial concentration of one reactant but keep the other reactant at saturation concentration?
the rate
What is k’?
the pseudo first-order rate constant
What is kcat?
how well the enzyme completes one cycle
kcat = Vmax/[E]t
What is Km?
How well the substrate and enzyme interact
what is kcat/Km?
Specificity constant: How fast does the enzyme turnover
Catalysts affect both the forward and reverse reaction. (T/F)
T
What are the common environmental conditions under which an enzyme (biological catalyst) performs?
Temperature (35-40 C)
pH (6-8)
What is the difference between cofactors and prosthetic groups?
prosthetic groups are tightly bound or covalently bound cofactors (small organic molecules)
The reaction rate is directly proportional to what?
The concentration of the reactants
Why are enzyme kinetics studied?
- understand effects of experimental conditions
- understand mechanism of enzyme
- investigation of metabolic pathways
- understand regulation of biochemical reactions (up or down regulation of catalyst)
- Describes its catalytic power, substrate affinity, and its response to inhibitors (helps design drugs)
What did Victor Henry do in 1903?
Proposed that an enzyme combines with its substrate molecule to form an ES complex as a necessary step in enzyme catalysis
What did Leonor Michaelis and Maud Menton do in 1913?
Expanded on the idea of Victory Henry into a general theory of enzyme actions.
How is steady state approximation (SSA) used to study enzymes?
It assumes the concentration of the intermediate is constant.
rate of ES = 0
If [S] is»_space;»> than km, what do we find?
km can be ignored in the Michaelis-Menton equation and the rate = vmax=k2[Eo]
If [S] is ««<
rate = (k2[E0][S])/km
If [S] = km, what do we find?
rate = Vmax/2
Vmax is commonly referred to as the what?
limiting rate
Is there a rate controlling step when [S] «<
no
Km is the substrate concentration when the limiting rate is what?
Half its value
What is a measure of affinity of enzyme for S and can be a measure for substrate affinity if k2
km
What does a low km indicate?
A fast formation of ES/tight ES complex (hard for it to dissociate)
What is the initial reaction velocity when the substrate concentration is very high compared to km?
Vmax
What is the maximum velocity independent enzyme concentration known as the catalytic constant (turnover number)?
kcat = Vmax/[Eo]=k2[Eo]/[Eo]=k2
What two things contribute to the maximum velocity limit?
- amount of enzyme
2. ability of enzyme (catalytic constant) kcat
What measures the affinity of an enzyme for a specific substrate?
kcat/km (enzyme efficiency or specificity constant)
The specificity constant is useful when what?
comparing primary substrate to other substrates (e.g. ethanol vs. propanol in alcohol dehydrogenase
Efficient enzymes have large what?
kcat/km (specificity constants)
large kcat and/or small km
What is considered the maximum efficiency of enzymes and is controlled by the diffusion limit of the substrate?
10^8 or 10^9 1/(M*s)
What is the rate at which the ES complex is formed?
kcat/km = (k1*k2)/(k-1+k2)=k1k2/k2=k1
Enzymes with efficiencies in the range of 10^8 to 10^9 (1/(M*s)) are said to have reached what?
Catalytic/kinetic perfection
How do we conduct kinetic experiments?
- Find out a reasonable amount of enzyme for the reaction
- Do an experiment with varying starting material and keeping the second substrate at a very high concentration
- measure product formed per second (plot [P] vs time)
- measure initial rate
- plot initial rate versus substrate concentration
What do you plot in a Lineweaver and Burk plot?
1/v vs 1/[S]
In a Lineweaver and Burk plot what is the y-intercept?
1/vmax
In a Lineweaver and Burk plot what is the x-intercept?
-1/km
In a Hanes-Wolf plot what do you plot?
[S]/v vs [S}
In a Hanes-Wolf plot what is the slope
1/vmax
In a Hanes-Wolf plot what is the x-intercept?
-km
What do you plot in an Eadie-Hofstee plot?
vo vs vo/[S]
What is the y-intercept of an Eadie-Hofstee plot?
vmax
What is the slope of an Eadie-Hofstee plot?
-km
What do you do to the Michaelis-Menton Equation to form the Lineweaver and Burk equation?
take the inverse
What do you do to the Lineweaver and Burk Equation to form the Hane-Wolf equation?
Multiply through with [S]
What do you do to the Michaelis-Menton Equation to form the Eadie-Hofstee plot?
rearrange the equation by:
1) multiply both sides by the denominator
2) distribute v0 on the left side
3) move the vokm term back to the right side
4) divide both sides by [S]
