Lecture 6 Information Flashcards
How do enzymes enhance the rate of catalysis?
stabilize the transition state
Transition state
a fleeting molecular moment in which events such as bond breakage, bond formation, and charge development are equally likely to revert to substrate or proceed to product
the transition state is transient
Why do enzymes bind to the transition state?
If enzymes binded to the ground state or lowered the energy of ES, the reaction would be less likely to occur
would take more energy to get to TS
activation energy
the difference between the energy levels of the ground state and the transition state
What types of reactions do enzymes normally catalyze?
slow, exergonic reactions
*reactions that would take place but are just too slow
Why are enzymes relatively large?
need to maintain structure of the active site
if enzymes were small, conformational changes would alter the active site more
binding energy
the total energy derived from enzyme-substrate interaction
where does binding energy come from?
1) free energy is released by forming many weak bonds and interactions between an enzyme and its substrate
2) weak interactions are optimized in the reaction transition state
how is binding energy used?
increase in free energy needed to reach the transition state is offset by the binding energy that is produced by enzyme-sustrate interactions in the transition state
binding energy functions as energy payment that lowers net activation energy and increases the speed of the reaction
does binding energy affect rate of reaction?
yes!
specificity
an enzyme’s ability to discriminate between a substrate and a competing molecule
where does an enzyme’s specificity come from?
the formation of many weak interactions between the enzyme and its specific substrate molecule
weak interactions spark both catalysis and specificity
examples of coenzymes
NAD and FAD
What are barriers binding energy can be used to overcome?
1) Entropy
2) Desolvation
3) Distortion
4) Proper alignment of the enzyme itself
Entropy and enzymes
substrates can be oriented correctly by the enzyme for reaction to occur
need to reduce entropy (by binding to enzyme) for the motion of the substrates to reduce and a reaction to occur
Desolvation and enzymes
enzyme-substrate interactions replace most or all of the hydrogen bonds with water that would otherwise impede the reaction
Induced fit
brings specific functional groups on the enzyme into the proper position to catalyze the reaction
the conformational change also permits formation of additional weak bonding interactions in the transition state
What is another way enzymes contribute to the overall catalytic mechanism besides binding energy?
covalent interactions between catalytic functional groups in the active site
Are covalent interactions between the enzyme and substrate permanent?
no
they are transient
transient
lasting for only a short period of time
impermanent
Types of covalent interactions between enzyme and substrate
1) General acid-base catalysis (donate/remove proton)
2) Covalent catalysis (create transient intermediate)
3) Metal Ion catalysis (help orient and stabilize the substrate; can mediate oxidation-reduction reactions)
Enzyme kinetics
determining the rate of a reaction and how it changes in response to changes in experimental parameters
Two steps of enzyme activity
E+S → ES (substrate binding step that usually occurs very fast)
ES –> E+P (slower second reaction that usually limits overall reaction)
What does K1 and K-1 refer to?
K1 is the forward rate of E+S → ES
K-1 is the reverse rate of E+S → ES
When is Vmax observed?
when virtually all the enzyme is present as the ES complex since need ES for second rate-limiting step to occur
Vmax
the maximum initial rate of the catalyzed reaction
Since ES –> E+P is generally the rate-limiting step, what does that tell us about the overall rate of the reaction?
the overall rate of the reaction depends on the amount of ES present
What happens at Vmax?
the enzyme is saturated with its substrate and further increases in [S] have no effect on rate
steady state
point in the reaction where [ES] remains approximately constant overtime
the steady state is normally achieved quickly
steady-state assumption
the initial rate of reaction reflects a state state in which [ES] is constant (the rate of the formation of ES is equal to the rate of its breakdown)
Michaelis-Menteen equation
a statement of the quantitative relationship between the initial velocity, the maximum velocity, the initial substrate concentration, and the Micahelis constant (Km)
When K2 is rate limiting (much slower than K1) what does Km reduce to?
the dissociation constant (K-1/K1)
can now determine the binding affinity of the enzyme
what is Kcat equivalent to?
the rate constant of the limiting step
Kcat
called the turnover number
equivalent to the number of substrate molecules converted to product in a given unit of time on a single enzyme molecule when the molecule is saturated with substrate
describes the ease/difficulty of catalysis
What happens at 1/2Vmax?
Km=S
As Km decreases…
binding affinity increases
As Km increases…
binding affinity decreases
specificity constant
kcat/km
allows us to compare the affinity of a substrate to bind to an enzyme and the turnover rate
Diffusion-controlled limit
perfect reaction when 2 molecules come in contact with each other and immediately react
cannot get any faster
When do enzymes reach the diffusion-controlled limit?
when kcat is 10^8-10^9 in power
enzyme inhibitors
molecules that interfere with catalysis, slowing or halting enzymatic reactions
Two broad classes of inhibitors
reversible and irreversible
competitive inhibitor
a type of reversible inhibitor
competes with the substrate for the active site of an enzyme. when the competitive inhibitor is in the active site, the substrate cannot bind
uncompetitive inhibitor
a type of reversible inhibitor
binds at a site distinct from the substrate and binds only to the ES complex
kinematic effects of competitive inhibitor
raises Km and does not effect Vmax
kinematic effects of uncompetitive inhibitor
Km decreases
Vmax decreases
mixed inhibitor
a type of reversible inhibitor
binds at a site distinct from the substrate active site, but it binds either to E or ES
kinematic effects of mixed inhibitor
affects both Km and Vmax
irrevisible inhibitors
bind covalently with or destroy a functional group on an enzyme that is essential for the enzyme’s activity or they form a super stable noncovalent interaction
transition-state analogs
stable molecules that resemble transition states
they bind to an enzyme more tightly than the substrate itself
resemble the transition-state structure of the normal enzyme
What is the main job of an enzyme?
to increase the rate of catalysis
Do enzymes change equilibrium point of a reaction?
No! Delta G stays the same since not changing the starting energies of the products or reactants
Why do we measure the initial rate of reaction Vo?
changes in [S] are small and [S] becomes a constant
Lineweaver-Burke plot
allows us to solve for the rate of an enzymatic reaction at infinite substrate concentrations
Where do enzymes have maximum binding affinity?
with the transition state
Why do we measure the initial velocities in enzyme kinematics?
at the initial velocities, we know the amount of substrate we just added
this allows us to have a constant [S]
Why do we not use K-2?
too slow to go backwards
How to make a Lineweaver Burke plot?
inver the Michaelis-Menton equation
graph 1/v versus 1/[s]
What is the y-intercept of lineweaver-burk?
1/Vmax
What is the x-intercept of lineweaver burk?
-1/Km
What is the slope of the lineweaver burk?
Km/Vmax
